Recombinant anti-Plasmodium falciparum antibodies

ABSTRACT

The present invention relates to a recombinant human antibody comprising an antibodysequence specific for the MSP-3 antigen of  Plasmodium falciparum . In particular, the invention relates to a recombinant human antibody which is specific for the MSP-3 194-257  antigen. The invention further relates to nucleic acid encoding such antibodies and to uses of these antibodies, in particular in the treatment or prophylaxis of malaria.

The present invention relates to recombinant antibodies against themalaria parasite, e.g. Plasmodium falciparum, to methods of producingsuch antibodies, and to uses and methods of use of such antibodies. Inparticular, the invention relates to antibodies against a specificantigen; MSP-3.

PRIOR ART

Every year more than 2.5 million people, mostly children, die ofmalaria.

The mortality and morbidity of this disease places a devastating burdenon the societies of the developing world. The United Nations hasestimated that the loss of production due to malaria in many Africancountries equals 5% of the gross national product.

Resistance of the malaria parasite (P. falciparum) to the existinganti-malarial drug chloroquine emerged in the sixties and has beenspreading since then. In addition, the malaria parasite has developedresistance to most other anti-malarial drugs over the past decade. Thisposes a major threat to public health. There is every reason to believethat the prevalence and degree of anti-malarial drug resistance willcontinue to increase. Furthermore, many anti-malaria drugs have beennotorious for their toxic side effects, e.g. mefloquin.

It has been previously shown that anti-parasitic activity from an immunedonor can be transferred by IgG into non-immune receivers (McGregor etal. 1963; McGregor 1964), and that such antibodies do not act directly,but act indirectly through a mechanism termed antibody-dependentcellular inhibition of growth (ADCI, Bouharoun et al. 1990). Thismechanism works preferentially with IgG1 and IgG3 (Bouharoun-Tayoun etal. 1992; Shi et al. 1999); (Aribot et al. 1996).

Using an ADCI assay, the merozoite surface antigen (MSP-3) waspreviously identified (Oeuvray et al. 1994). Affinity purified humanpolyclonal anti-MSP-3 antibodies have also been used in a mouse model(Badell et al. 2000).

There are a number of difficulties in using the antibodies describedabove, i.e. those purified from an infected host in the prevention ortreatment of malaria. From a practical point of view it is highlyunlikely that serum from infected individuals would ever be approved formedical use (for obvious safety reasons). Neither would sufficientamounts of antibodies be available.

In principle, recombinant human antibodies with appropriate specificityand activity were desirable. However, there have been difficulties inthe art in developing these. For example, Seehaus et al. (1992)describes problems associated with the occurrence of deletion mutants inphage display protocols, which, despite being undesirable, tend toreplicate faster than “intact” phages and so become over-represented.

DISCLOSURE OF THE INVENTION

The present inventors have, for the first time, developed recombinanthuman antibodies specific for the MSP-3 antigen. These antibodies areable to passively induce naturally occurring non-sterile malariaimmunity (termed premunition) and exert their effect via the ADCImechanism. Additionally, the recombinant antibodies may stimulatephagocytosis of infected erythrocytes and free parasites. Yet anothermechanism of action of the antibodies may be direct interference withthe function of the MSP-3 and/or the MSP-6 molecules by binding to theepitope.

Specifically, the inventors generated a phage display system based onthe amplification of genes from the human peripheral blood leukocytes of13 malaria immune individuals which encode variable regions (V) ofantibody. These genes were isolated by the polymerase chain reaction(PCR).

Following amplification of the V region genes, these genes were clonedinto a compatible phagemid vector, which directs expression of antibodyfragments. The resultant phage display library was screened using afragment of the merozoite surface protein, MSP-3; MSP-3₁₉₄₋₂₅₇ (Oeuvrayet al. 1994).

Specifically, the inventors developed a new panning strategy to screenthe library. In this method, biotinylated MSP-3₁₉₄₋₂₅₇ antigen wascoupled to streptavidin coated polystyrene beads (Dynabeads), andincubated with the phage library.

The inventors thought that good binders would be present in very lowfrequency in the initial stock. To recover a high proportion of raregood binders, a high initial concentration of antigen was thought to beneeded. However, in subsequent steps the good binders were assumed to bepresent with increasing frequency. So, the inventors reduced the amountof antigen used in each panning. This avoided the survival of mediocrebinders.

The inventors strategy involved reduction of the number of antigencoated beads in subsequent pannings. In this way, both the amount ofantigen and the area of matrix (the beads) are reduced concomitantly.

This concomitant reduction of the surface area of the matrix and theamount of antigen avoids a selective advantage of deletion mutantssurviving by non-specific adherence to matrix.

The selection procedure resulted in isolation of three distinct clonesdesignated RAM1, RAM2 and RAM3. RAM1 was predominant in the 376phage-clones examined. Epitope mapping performed on these clonesrevealed the relationship between these clones.

The reactivity of the clones with native malaria antigen wasdemonstrated by immunoblotting, immunofluorescence and flow cytometry.

The RAM1, RAM2 and RAM3 clones were produced as intact human IgG1 andintact IgG3 antibody in eukaryotic CHO cells. These antibodies have beenpurified to more than 99% purity.

Aspects of the Invention

Accordingly, in a first aspect the present invention provides arecombinant human antibody comprising an antibody sequence specific forthe MSP-3 antigen of Plasmodium falciparum.

An antibody is specific for a particular antigen if it binds thatparticular antigen in preference to other antigens. In particular, theantibody may not show any significant binding to molecules other thanthat particular antigen, and specificity may be defined by thedifference in affinity between the target antigen and other non-targetantigens. An antibody may also be specific for a particular epitopewhich may be carried by a number of antigens, in which case the antibodywill be able to bind to the various antigens carrying that epitope. Forexample, specific binding may exist when the dissociation constant for adimeric complex of antibody and antigen is 1 μM, preferably 100 nM andmost preferably 1 nM or lower.

The recombinant human antibody of the invention is preferably specificfor the C-terminus of the MSP3 antigen, preferably the MSP-3₁₉₄₋₂₅₇antigen.

The recombinant human antibody of the invention may be specific for theepitope having the amino acid sequence ILGWEFGGGVP (SEQ ID NO: 10) whichcorresponds to residues 220-230 of the MSP-3 antigen. This sequence isalso conserved in other antigens such as the MSP-6 antigen of Plasmodiumfalciparum (Trucco et al. 2001).

The recombinant human antibody may comprise a CDR sequence selectedfrom: CDR1 of RAM1 VH; CDR2 of RAM1 VH; CDR3 of RAM1 VH; CDR1 of RAM1VK; CDR2 of RAM1 VK; CDR3 of RAM1 VK; CDR1 of RAM2 VH; CDR2 of RAM2 VH;CDR3 of RAM2 VH; CDR1 of RAM2 VK; CDR2 of RAM2 VK; CDR3 of RAM2 VK; CDR1of RAM3 VH; CDR2 of RAM3 VH; CDR3 of RAM3 VH; CDR1 of RAM3 VK; CDR2 ofRAM3 VK; or CDR3 of RAM3 VK, having the sequences shown in FIG. 6 (theCDRs are marked and are underlined on FIG. 6).

Preferably, the antibody comprises the CDR3 from a heavy chain, that isa CDR selected from: CDR3 of RAM1 VH; CDR3 of RAM2 VH; or CDR3 of RAM3VH, having the sequences as shown in FIG. 6.

More preferably, the antibody comprises two or more CDRs shown in FIG.6, i.e. two or more CDRs selected from CDR1 of RAM1 VH; CDR2 of RAM1 VH;CDR3 of RAM1 VH; CDR1 of RAM1 VK; CDR2 of RAM1 VK; CDR3 of RAM1 VK; CDR1of RAM2 VH; CDR2 of RAM2 VH; CDR3 of RAM2 VH; CDR1 of RAM2 VK; CDR2 ofRAM2 VK; CDR3 of RAM2 VK; CDR1 of RAM3 VH; CDR2 of RAM3 VH; CDR3 of RAM3VH; CDR1 of RAM3 VK; CDR2 of RAM3 VK; or CDR3 of RAM3 VK having thesequence shown in FIG. 6.

The antibody may comprise any one of CDR1, CDR2, and CDR3 of a heavychain as shown in FIG. 6 and any one CDR1, CDR2 and CDR3 of a lightchain as shown in FIG. 6. For example the antibody may comprise CDR3 ofa heavy chain and CDR1 of a light chain. Or, the antibody may comprisetwo or more CDRs from a heavy chain, or two or more CDRs from a lightchain as shown on FIG. 6.

Most preferred is that the antibody comprises CDR1, CDR2 and CDR3 of alight chain as shown in FIG. 6, and CDR1, CDR2 and CDR3 of a heavy chainas shown in FIG. 6.

It is preferred that, where the antibody comprise two or more CDRs, thetwo or more CDRs are from the same RAM clone, that is two or more CDRsfrom RAM1; two or more CDRs from RAM2 or two or more CDRs from RAM3.

It is most preferred that the antibody comprises the CDR1, CDR2, CDR3 ofthe light chain and CDR1, CDR2 and CDR3 of the heavy chain, wherein allsix CDRs are from the same RAM clone, e.g. all six CDRs are from RAM1.

Although it is preferred that within a single antibody the CDRs are froma single RAM clone, e.g. RAM1, RAM2 or RAM3, any combination ispossible, that is where the antibody comprises two or more CDRs asdisclosed herein, a first CDR may be from any one of RAM1; RAM2 and RAM3and the second CDR may be from any of RAM1; RAM2 or RAM3. To producecombinations in which CDRs derive from different RAM clones, CDRshuffling (Jirholt P, Ohlin M, Borrebaeck Calif., Soderlind E. (1998).Exploiting sequence space: shuffling in vivo formed complementaritydetermining regions into a master framework. Gene 1998 Jul 30; 215 (2):417-6) or DNA shuffling may be used (Crameri et al., 1996).

Preferably, the recombinant human antibody may comprise a V_(H) domain(Variable domain of the heavy chain) selected from RAM1 VH; RAM2 VH orRAM3 VH having the amino acid sequence shown in FIG. 6 (the VH aminoacid sequences are shown as the top line for each RAM clone in FIG. 6).

Preferably, the recombinant human antibody has a V_(L) domain (Variabledomain of the light chain) selected from RAM1 VK; RAM2 VK; or RAM3 VKhaving the amino acid sequence of RAM1, RAM2 or RAM3 shown in FIG. 6(the VK amino acid sequences are shown as the bottom line for each RAMclone in FIG. 6). Any light chain may be combined with any heavy chain,e.g., by light and heavy chain shuffling (Marks et al. 1992).

However, it is preferred that the recombinant human antibody has theV_(L) domain of RAM 1 VK and the VH domain of RAM1 VH; or the VL domainof RAM2 VK and the VH domain of RAM2 VH; or the VL domain of RAM3 VK andthe VH domain of RAM3 VK, as shown in FIG. 6.

The variable heavy chain regions discussed above may be combined withany suitable constant region, including the constant region of gamma 1,gamma 2, gamma 3, gamma 4, my, alfa 1, alfa 2, delta or epsilon isotypesas well as any artificial constant region. It is preferred that therecombinant human antibody comprises a constant region of the gamma 1,gamma 2, gamma 3, or gamma 4 isotypes, to form an IgG molecule.

More preferably, the recombinant human antibody comprises a constantregion of the gamma 1 or the gamma 3 isotypes, to form an IgG1 or IgG3isotype.

For each isotype, any subclass and any allotype may be used. It isdesirable that antibodies for therapeutic use should have as littleimmunogenic effect in the recipient as possible. Therefore it isdesirable that the allotype of a therapeutic antibody is one which therecipient normally expresses. Therefore common allotypes are preferred.Preferred IgG1 and IgG3 allotypes include G1m(a,z) and G3m(b). Furtherpreferred allotypes include G1m(f), G3m(c3c5), G3m(c3) and G3m(s).

Alternatively, other molecules capable of eliciting the desired effectorfunctions or anti-parasitic effects may be coupled to theantigen-binding region of the antibodies, e.g. enzymes withanti-parasitic effects. For example, the use of such antibodies mayconfer target selectivity to an otherwise toxic drug or substance.

Preferably the antibodies of the present invention are capable ofmediating antibody-dependent cellular inhibition (ADCI) of P. falciparumin vitro, resulting in P. falciparum killing by the methods describedherein, or known in the art (e.g. Bouharoun-Tayoun et al., 1995).

Preferably the antibodies are capable of inducing an antigen specificdecrease in P. falciparum parasitemia in a suitable immunocompromisedanimal model grafted with parasitised human red blood cells, e.g. the P.F.-HuRBC-BXN model described herein, or the mouse models described byBadell et al. (1995; 2000). In some embodiments a fast decrease isachieved.

Preferably the antibodies of the present invention are capable ofclearing P. falciparum parasitemia from such an animal model.

In some embodiments, the antibodies of the present invention will exerteffects either in vivo, or in vitro comparable to those obtained withpolyclonal antibodies purified from immune human donors, e.g. total IgG,or antibodies affinity purified on MSP3 antigens, such as full length orC-terminal recombinant MSP3 protein, or the MSP3b peptide describedherein. In some embodiments the effects are exerted in vivo and invitro. In some embodiments the recombinant antibodies provide the samedegree of P. falciparum killing as the antibodies obtained from donors.Thus some embodiments of the invention will provide a profoundbiological effect on P. falciparum under both in vivo and in vitroconditions.

The recombinant human antibodies of the invention may be produced byexpression from a suitable nucleic acid molecule.

In addition to an antibody sequence, the antibody may comprise otheramino acids, e.g. to impart to the molecule another functionalcharacteristic in addition to ability to bind antigen. For example, thespecific binding member may comprise a label, or enzyme and so on (asdiscussed in more detail below).

In addition to the complete antibody, fragments of the antibody may alsohave the ability to bind the appropriate antigen (such as MSP-3), andare therefore also encompassed by the invention. For example, it hasbeen shown that fragments of a whole antibody can perform the functionof binding antigens. Examples of binding fragments are (i) the Fabfragment consisting of VL, VH, CL and CH1 domains; (ii) the Fv fragmentconsisting of the VL and VH domains of a single antibody; (iii) isolatedCDR regions; (iv) F(ab′)2 fragments, a bivalent fragment comprising twolinked Fab fragments (v) single chain Fv molecules (scFv), wherein a VHdomain and a VL domain are linked by a peptide linker which allows thetwo domains to associate to form an antigen binding site (Bird et al,Science, 242, 423-426, 1988; Huston et al, PNAS USA, 85, 5879-5883,1988); (vi) bispecific single chain Fv dimers (PCT/US92/09965) and (vii)“diabodies”, multivalent or multispecific fragments constructed by genefusion (WO94/13804; P. Holliger et al, Proc. Natl. Acad. Sci. USA 906444-6448, 1993). Fv, scFv or diabody molecules may be stabilised by theincorporation of disulphide bridges linking the VH and VL domains (Y.Reiter et al, Nature Biotech, 14, 1239-1245, 1996). Minibodiescomprising a scFv joined to a CH3 domain may also be made (S. Hu et al,Cancer Res., 56, 3055-3061, 1996).

Accordingly, in a further aspect the present invention provides anisolated nucleic acid molecule comprising a nucleic acid sequenceencoding a recombinant human antibody of the first aspect.

Such a nucleic acid molecule may be in the form of a recombinant andpreferably replicable vector.

Such a ‘vector’ may be any plasmid, cosmid, or phage in double or singlestranded linear or circular form which may or may not be selftransmissible or mobilizable, and which can transform prokaryotic oreukaryotic host either by integration into the cellular genome or existextrachromosomally (e.g. autonomous replicating plasmid with an originof replication). Any suitable host may be used, including bacteria, e.g.archaebacteria, plants, plant cells, fungi or animal cells.

Generally speaking, those skilled in the art are well able to constructvectors and design protocols for recombinant gene expression. Forfurther details see, for example, Molecular Cloning: a LaboratoryManual: 2nd edition, Sambrook et al, 1989, Cold Spring Harbor LaboratoryPress (or later editions of this work)and Current Protocols in MolecularBiology, Second Edition, Ausubel et al. eds., John Wiley & Sons, 1992,which are incorporated herein by reference.

Preferred vectors include the plasmids pLNOH2 or pLNOK which aredescribed in Norderhaug et al. 1997 (and discussed in more detaillater). Other suitable vectors for expresssion of antibodies aredescribed in Sanna et al. 1999; Persic et al. 1997; Walls et al. 1993.

Plasmids pLNOH2 and pLNOK may be expressed by any suitable cell type,e.g. mammalian, yeast, insect or plant cells, especially mammalian cellssuch as BHK, CHO, or COS cells, using methods which are standard in theart. CHO or BHK cells are most preferred.

It is preferred that the antibody is secreted to the media by the cellfrom which it is expressed.

Further aspects of the invention relate to: a method of expressing in ahost cell an antibody as described herein from a nucleic acid moleculedescribed herein; a host cell capable of expressing an antibody asdescribed herein in appropriate culture conditions for producing saidantibody; a method of producing an antibody comprising culturing such ahost cell under appropriate conditions, which method may furthercomprise isolating said antibody from the cell culture, and which methodmay further comprise admixing the isolated antibody with a suitablefurther component (which may, for example, be another antibody or anexcipient or carrier).

Mammalian cells may be transfected by any suitable technique such aslipofection. One suitable method is described in the Examples.Alternatively, standard calcium phosphate transfection orelectroporation may be used, which is well understood by the skilledperson.

The recombinant antibodies produced from these expression systems andnucleic acid molecules of the invention are preferably provided in asubstantially pure or homogeneous form. Recombinant antibodies may bepurified by any suitable method such as ammonium sulphate precipitation,preferably followed by purification using a DEAE Sepaharose column andan ABx column (Baker Bond). This may optionally be followed by a gelfiltration step, e.g. using Superdex200 .

In a further aspect of the invention, there is provided a method forscreening a phage display library, comprising the following steps:

-   -   (i) attaching a target molecule (such as an antigen) to a bead        using a suitable linker;    -   (ii) admixing a first amount of such beads with attached target        molecule with a first population of phage from a phage display        library (which library expresses sequences (such as antibody        sequences) which may bind to the target molecule;    -   (iii) selecting from the first population of phage a second        population of phage, wherein the second population of phage is        enriched in phage which bind to the target molecule;    -   (iv) admixing said second population of phage with a second        amount of beads, wherein said second amount of beads is smaller        than said first amount;    -   (v) selecting from said second population of phage a third        population of phage, wherein said third population of phage        enriched in phage which bind to the target molecule.

Steps (iv) and (v) may be repeated to produce fourth and fifthpopulations.

Using this method, the amount of target molecule and area of bindingmatrix (total bead surface area) are reduced concomitantly.

It is preferred that the target molecule is an antigen and the phagedisplay library contains antibody sequences.

A suitable linker for attachment of the target molecule to the bead isan avidin-biotin linker.

A suitable reduction between the first and second amounts of beads is areduction by a factor of 10. For example, where the first amount ofbeads is 7×10⁵ the second amount of beads may be 70000. If steps (iv) to(v) are repeated then a third amount of beads may be around 7000.

Other reductions in the amount of beads are also possible and can bevaried by the skilled person. The reductions do not need to be the samethroughout the method. For example, the reduction between first andsecond amounts may be by a factor of 10, and between the second andthird amounts may be a factor of 5. In another example, the reduction inbetween first and second amounts may be by a factor of 2 and betweensecond and third amounts may be by a factor of 5.

The method of screening a phage display library may (where the phagedisplay library contains antibody sequences) be followed by the furtherstep of producing a complete antibody. Producing a complete antibody maycomprise insertion of the antibody sequences isolated from the phagedisplay library into a suitable vector (such as pLNOH2 and pLNOK, asdescribed herein) and expressing the antibody from that vector in anappropriate cell type.

Antibodies produced or producible by such a method represent a furtheraspect of the invention.

Uses of Antibodies

The antibodies disclosed herein can be used for treatment or forprevention of a malarial disease.

The term “malarial disease” includes clinical, e.g. clinical symptomaticinfection, clinical asymptomatic infection and cerebral malaria.

Accordingly, in a further aspect the present invention providesrecombinant human antibodies disclosed herein for use in the treatmentor prophylaxis of a malarial disease.

The invention further encompasses the use of recombinant humanantibodies disclosed herein in the manufacture of a medicament (e.g. avaccine) for the treatment or prophylaxis of malaria. The medicament mayfurther comprise a suitable excipient or carrier.

Suitable excipient, carriers, buffers, stabilises are well known tothose skilled in the art. The precise nature of the carrier or othermaterial will depend on the route of administration, which may be oral,or by injection, e.g. cutaneous, subcutaneous or intravenous.

Pharmaceutical compositions for oral administration may be in tablet,capsule, powder or liquid form. A tablet may include a solid carriersuch as gelatin or an adjuvant. Liquid pharmaceutical compositionsgenerally include a liquid carrier such as water, petroleum, animal orvegetable oils, mineral oil or synthetic oil. Physiological salinesolution, dextrose or other saccharide solution or glycols such asethylene glycol, propylene glycol or polyethylene glycol may beincluded.

For intravenous, cutaneous or subcutaneous injection, or injection atthe site of affliction, the active ingredient will be in the form of aparenterally acceptable aqueous solution which is pyrogen-free and hassuitable pH, isotonicity and stability. Those of relevant skill in theart are well able to prepare suitable solutions using, for example,isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection,or Lactated Ringer's Injection. Preservatives, stabilisers, buffers,antioxidants and/or other additives may be included, as required.

Methods of treatment or prophylaxis of malaria form further aspects ofthe invention, such methods may comprise administering a recombinanthuman antibody as described herein to an individual.

In the above methods and uses, the recombinant antibodies may beadministered to the individual in any of the following ways: intravenousinjection; intramuscular injection; sub-dermal injection; oraladministration e.g. using capsules to protect the antibody duringpassage to the stomach; administration in toothpaste (whereby theantibody is transferred to the circulation by the abrasive effects inthe oral mucosa); administration by the use of occlusive plasters orskin ointment for penetration through the skin; administration throughthe nasal mucosa by delivery with a spray; and rectal administration.

Suitable doses for treatment may be 10 μg to 10 mg of antibody per kgbody weight or less depending on the affinity of the antibody,preferably 1 mg to 10 mg, more preferably 0.1 mg to 3.5 mg, morepreferably 1.5 mg to 3.5 mg, most preferably 2 mg antibody per kg bodyweight.

Suitable doses for prevention may be smaller due to the lower load ofparasites to be eliminated, such as 0.1 μg to 3 mg, preferably 0.1 mg to2 mg, more preferably 0.5 mg to 1.5 mg, more preferably 0.75 mg to 1 mgmost preferably 1 mg antibody per kg body weight.

Suitable administration schemes may be based upon an in vivo half-lifefor the antibodies of the IgG1 class of around to 23 days and an in vivohalf-life for the IgG3 class of around 6 days.

Alternatively, or additionally, the nucleic acid molecule encoding theantibody may be used in the treatment or prevention of malarial disease.In this way the DNA is injected into the individual and the antibody isproduced endogenously in that individual.

Accordingly, further aspects of the invention relate to: the nucleicacid molecules described herein for use in the treatment or prophylaxisof a malarial disease; the use of nucleic acid molecules describedherein in the manufacture of a medicament (e.g. a vaccine) for thetreatment or prophylaxis of malaria; and methods of treatment orprophylaxis of malaria comprising administering a nucleic acid moleculeas described herein to an individual.

Suitable vectors for the administration of nucleic acid molecules to anindividual include adenovirus and adeno-associated virus, and retrovirusvectors, in addition to other particles that introduce DNA into cells,such as liposomes.

A further aspect of the present invention provides a method whichcomprises causing or allowing binding of an antibody as provided hereinto a target antigen.

Such binding may take place in vivo, e.g. following administration of anantibody, or nucleic acid encoding a antibody, or it may take place invitro, for example in ELISA, Western blotting, immunocytochemistry,immuno-precipitation or affinity chromatography.

In vivo, this may be useful for vaccine where the antibodies disclosedherein may serve as an adjuvant to enhance the endogenous immuneresponse. Alternatively, the antibodies disclosed herein may serve astransport molecule facilitating the uptake of the MSP-3 antigen, e.g.from the intestinal tract (for this latter function IgA is preferred).When the antibodies disclosed herein are administered in vivo duringnatural infection with P. falciparum a protective endogenous immuneresponse is facilitated, i.e. passive immunization combined withinfection facilitates active immunization (Zhang et al. 2002; Manca etal. 1988).

The amount or extent of binding of specific binding member to a targetantigen may be determined. Quantitation may be related to the amount ofthe antigen in a test sample, which may be of diagnostic interest.

The reactivities of specific binding members on a sample may bedetermined by any appropriate means. Radioimmunoassay (RIA) is onepossibility. Radioactive labelled antigen is mixed with unlabelledantigen (the test sample) and allowed to bind to the specific bindingmember. Bound antigen is physically separated from unbound antigen andthe amount of radioactive antigen bound to the specific binding memberdetermined. The more antigen there is in the test sample the lessradioactive antigen will bind to the specific binding member. A bindingassay may also be used with non-radioactive antigen, using antigen or ananalogue linked to a reporter molecule. The reporter molecule may be afluorochrome, phosphor or laser dye with spectrally isolated absorptionor emission characteristics. Suitable fluorochromes include fluorescein,rhodamine, phycoerythrin and Texas Red. Suitable chromogenic dyesinclude diaminobenzidine.

The present invention also provides the use of a specific binding memberas above for measuring antigen levels in a competition assay, that is tosay a method of measuring the level of antigen in a sample by employinga specific binding member as provided by the present invention in acompetition assay. This may be where the physical separation of boundfrom unbound antigen is not required. Linking a reporter molecule to thespecific binding member so that a physical or optical change occurs onbinding is one possibility. The reporter molecule may directly orindirectly generate detectable, and preferably measurable, signals. Thelinkage of reporter molecules may be directly or indirectly, covalently,e.g. via a peptide bond or non-covalently. Linkage via a peptide bondmay be as a result of recombinant expression of a gene fusion encodingantibody and reporter molecule.

Thus a further aspect of the invention is a method of diagnosis ofmalaria. Such a method may comprise taking a sample of bodily fluid froman individual, contacting the sample with an antibody as describedherein and determining the binding of that antibody to the sample,thereby determining the presence or absence of a target antigen in thesample. Thus the binding of an anti-MSP-3 antibody described hereinwould show the presence of the MSP-3 antigen in the sample.

In addition, antibodies according to the invention may be used inmethods to assist in the identification or production of molecules withanti-parasitic, e.g. anti Plasmodium falciparum activity. In this way,antibodies may be used to guide the selection of novel antibodiesagainst the MSP-3 antigen or cross reactive antigen (Ohlin et al, 1996;Jirholt et al, 1998), or the antibodies may be used to identify thesingle components in the protective mechanisms responsible for theanti-parasitic effects and to identify other substances enhancing orinterfering with these effects.

The invention will now be described in detail with reference to thefollowing figures and examples, which are non-limiting.

BRIEF DESCRIPTION OF THE DRAWINGS AND TABLE I

FIG. 1 shows ELISA reactivity with various truncated versions of MSP-3.Fab-ΔpIII fragments produced from the three distinct clones wereanalysed in ELISA. Truncated recombinantly produced, MSP-3₂₂₋₂₅₇ (blackbars) , MSP-3₁₉₄₋₂₅₇ (grey bars) were used as coating antigens inseparate analyses. An ELISA for detection of Fab fragments (Anti-Fab)was included to ensure use of comparable amounts of the various Fabfragments (white bars). Background was measured as reactivity with thecontrol Fab against HibCP and as reactivity with buffer. Reactivitiesare indicated as OD₄₀₅-OD₄₉₀. Bars indicated median values anderror-bars indicated 2 times standard deviation of triplicates.

FIG. 2 shows results of the antigen competition experiments. Fab-ΔpIIIfrom the three distinct clones were competed with soluble panningantigen in ELISA. All three clones were susceptible to competition withsoluble MSP-3₁₉₄₋₂₅₇. A constant amount of Fab-ΔpIII produced from eachof the three distinct clones was mixed with varying amounts ofcompetition antigen, MSP-3₁₉₄₋₂₅₇, and applied to ELISA wells coatedwith the same antigen. Percent reactivity in the ELISA was calculatedtaking the reactivity with no antigen added as 100%.

FIG. 3 shows a schematic representation of the entire MSP-3 antigen(top, MSP-3₁₋₃₈₀) and the relationship of the truncated antigens(MSP-3₂₂₋₂₅₇, MSP-3₁₉₄₋₂₅₇, (SEQ ID NO: 7), MSP-3₁₉₀₋₂₁₇ (SEQ ID NO: 8)and MSP-3₂₁₁₋₂₃₇ (SEQ ID NO: 9)used). The numbering above has beenassigned according to the P. falciparum clone D10, sequenced by McCollet al. 1994 (Genbank accession number L07944). As this numbering may bedifferent when aligned with other clones, the MSP-3₁₉₄₋₂₅₇ (Oeuvray etal. 1994, accession number AF024624) amino acid sequence has beenwritten in the figure.

The intact antigen comprises 12 heptad repeats, equally distributed intothe three heptad repeat regions (HELIX 1, HELIX 2, HELIX 3). Analyses ofthe sequence of the regions suggest that they have an amphipaticα-helical secondary structure and in the presence of all three regionscan obtain the structure of a coiled-coil three-stranded helical bundle(McColl et al. 1994; Mulhern et al. 1995). The helix regions and signalpeptide (SIGNAL) are indicated as boxes at the top of the drawing. Thedimorphic areas described by Huber et al. (1997) are indicated with grayshading. The MSP-3₁₉₀₋₂₁₇ peptide represents the Helix 3 sequence of theK1 clone (McColl et al. 1997, accession number U08851).

The motif represented by amino acid residues 220-230, ILGWEFGGGVP (SEQID NO: 10), is indicated by oval figures. This motif is present in MSP-3as well as in the other Plasmodium falciparum antigen MSP-6 (Trucco etal. 2001).

FIG. 4 shows the results of the flow cytometry analyses of thereactivity of recombinant antibodies with schizonts. Panel A1illustrates forward scatter and side scatter for ethanol-fixed and—permeabilized infected red blood cells, and panel A2 illustrates thesame parameters for similarly treated non-infected red blood cells. Thegating was placed to include the majority of the infected cells. Thegating was used for the histograms described below.

Panel B1 to 4 illustrates histograms of various recombinant anti-malariaantibodies compared with the control antibody directed against HibCPantigen (grey shaded curve). The control histogram has 2% of the eventsunder the bar designated M1 in panel B1.

B1 shows RAM1 in the Fab-ΔpIII format. The bar covers 76% of the eventsregistered.

B2 shows RAM2 in the Fab-ΔpIII format. The bar covers 28% of the eventsregistered.

B3 shows RAM3 in the Fab-ΔpIII format. The bar covers 4% of the eventsregistered.

B4 shows RAM1 in the IgG1 format. The bar covers 47% of the eventsregistered. The control in the IgG1 format has 4% of the events underthe bar.

Schizonts were purified from asynchronous P. falciparum culture.Fixation and permeabilization was carried out with ethanol. Primaryreaction with recombinant antibody was carried out over night and theFITC-labeled secondary antibody was incubated with the cells for 30minutes.

FIG. 5 shows a schematic representation of the panning procedure. Threeparallel series (A to C) of panning were carried out for 4 roundsdesignated 1 to 4. The number of antigen-coated beads used in each roundwas reduced by a constant reduction factor through the study. The numberof beads used for each stage is indicated in the figure.

FIG. 6 shows the deduced amino acid sequences of the three clones. Theentire VH and VK sequences of each clone, RAM1(SEQ ID NOS 1-2,respectively, in order or appearance), RAM2 (SEQ ID NOS 3-4,respectively, in order or appearance) and RAM3 (SEQ ID NOS 5-6,respectively, in order or appearance) are shown and boxed. Amino acidresidues in the CDR regions are underlined. We have chosen to show theKabat CDR definitions (Kabat et al. 1991).

FIG. 7 shows the immunoblotting of P. falciparum clone 3D7 schizont andmerozoite proteins reacted with RAM1, RAM2 and RAM3 produced as IgG1.All three lanes show a blotting of proteins from purified schizontsseparated under reducing conditions on a 4-12% gradient gel. RAM1 IgG1was reacted with the blotting in lane 1, RAM2 IgG1 was reacted with theblotting in lane 2 and RAM3 IgG1 was reacted with the blotting in lane3.

RAM1 reacts with two proteins of molecular weights 51 and 53 kD,respectively.

RAM2 reacts with two proteins of approximately relative molecularweights of 64 kD and 14 kD, respectively.

RAM3 reacts with a protein of approximately relative molecular weight of64 kD identical to the protein recognized by RAM2.

FIG. 8 shows Immunofluorescence microscopy. Panels representimmunofluorescence microscopy of fixed culture of P. falciparum clone3D7 incubated with FITC-conjugated RAM1IgG1, or RAM2 IgG1 or RAM3 IgG1followed by a FITC conjugated secondary anti-human Fab. Dots in the A(left) panels indicate red cells carrying antigen reactive with RAM1,RAM2 or RAM3. The B (right) panels represents the same slides stainedfor DNA with propidium iodide. Large dots in B panels indicate red cellsinfected with late stage schizonts. By comparing the location of thedots in the A and B panels it is observed that the larger dots in Bpanels are duplicated in the left panels. Small dots in the B panels arenot duplicated in A panels. This demonstrates that late-stage schizontsharbouring merozoites do display the antigen reactive with RAM1, RAM2 orRAM3 whereas early stages do not.

Asynchronous in vitro P. falciparum culture was air-dryed on a slide,fixed with acetone and reacted with FITC-conjugated RAM1 IgG1, orreacted with RAM2 IgG1 or RAM3 IgG1 followed by FITC-conjugatedanti-human Fab. No reactivity was detectable with glutaraldehydefixation.

FIG. 9 shows the ELISA reactivity of RAM1 IgG1, RAM2 IgG1 and RAM3 IgG1with the synthetic peptides MSP-3₁₉₀₋₂₁₇ and MSP-3₂₁₁₋₂₃₇. Also thelarger recombinant MSP-3₁₉₄₋₂₅₇ has been included. Error bars represent2 times standard deviation of duplicates.

FIG. 10 shows the results from the PEPSCAN screening. The entiresequence MSP-3₁₉₄₋₂₅₇ was covered by synthesis of 34 sets of 14-mer and15-mer peptides comprising contiguous amino acid sequences (SEQ ID NOS11-78, respectively, in order or appearance). The two peptides werelinked by a proprietary linker substituting two amino acid residues onthe location of the linker (PEPSCAN). Thus one single set of peptidescovers a stretch of 31 amino acid residues with two amino acid residuesreplaced in positions 15 and 16 by the proprietary linker residues. Thelinked peptides were furthermore chemically coupled to the matrix toallow washing and repeated use. The overlap between two adjacent sets ofpeptides was two amino acid residues. This collection of peptides wasexamined for reactivity with RAM1 and RAM2 produced as IgG1. RAM2 reactswith the amino-terminal, the middle and the carboxy-terminal part of theantigen but yields the highest reactivity with the middle part. RAM1reacts only weakly with the peptides produced by this method. RAM3 wastested as Fab-ΔpIII in PEPSCAN without response.

FIG. 11 shows the inhibitory effect of immune serum on the binding ofRAM1 IgG1 to MSP-3₁₉₄₋₂₅₇. The FITC-conjugated RAM1 IgG1 antibody wasapplied to ELISA wells coated with MSP-3₁₉₄₋₂₅₇ were pre-incubated fortwo hours with dilutions of immune or negative control serum and then aconstant amount of FITC-conjugated RAM1 IgG1antibody was applied to eachwell. Immune serum blocked the binding of RAM1.

FIG. 12 Construction of expression vectors. The V-regions of theplasmids can be exchanged by cutting with BsmI in the 5′ end and bycutting with HpaI or BsiWI or HindIII in the 3′ end. Before ligation ofvector and V region, the V regions of RAM1, RAM2 or RAM3 need to be cutwith the same restriction enzymes as the vectors. VH regions are thenligated to pLNOH2 and VK regions are ligated to pLNOK. The constantregion of pLNOH2 can be exchanged by cutting with BamHI and HindIII,isolating the vector part and introducing (by ligation) another constantregion cut with the same enzymes.

In order to produce a complete antibody the plasmids (both pLNOH2 andpLNOK) are co-transfected into CHO cells. The V and C-regions areassembled by mRNA splicing. Alternatively the genes can be assembledinto one vector thus harbouring the VH plus CH and VL plus CL. Thisinsures that the two genes are present in equimolar numbers.

Abbreviations: CMV=CMV promoter, L=leader, VH=variable heavy,VK=variable kappa, CH=constant heavy chain gene, CK=constant kappa chaingene, BGHpA=Bovine Growth Hormone polyadenylation site, f1=f1 origin,SV40ori=SV40 origin/promoter Neo=gene for neomycin resistance, Amp=genefor ampicillin resistance

FIG. 13 Antibody competition. Fab fragments from clone RAM3 wereproduced in two versions. One version was fusion protein tagged with atruncated phage protein, ΔpIII, designated Fab-ΔpIII, and anotherversion was normal Fab fragments without the ΔpIII tag. The ΔpIII-tag isreadily detected using an antibody directed to the phage pIII protein.Thus Fab without ΔpIII can be present as competitor but only Fab-ΔpIIIwill be detected.

Amounts of RAM1, RAM2 and RAM3 Fab-ΔpIII yielding OD₄₀₅ of approximately1 in ELISA on a coating of MSP-3₁₉₄₋₂₅₇ were used. The binding of theFab-ΔpIII was then competed by the addition of increasing amounts ofcompetitor, RAM3 without ΔpIII. This figure shows that Fab RAM3 is ableto compete Fab-ΔpIII RAM1 to the same extent as it competes Fab-ΔpIIIRAM2. This demonstrates that binding of the clones RAM1 and RAM2 isdependent on the epitope used by clone RAM3. The lower competitiveeffect of RAM3 on the binding of Fab-ΔpIII RAM3 could be explained by ahigher affinity of RAM3 as compared to RAM1 and RAM2. The effect of thiswould be that the competitive effect of RAM3 towards RAM1 and RAM2 wouldbe larger than towards RAM3 itself.

FIG. 14 shows the organization of heavy and light chain genes in pLNOH2and pLNOK, respectively, and the resulting antibody structure. The partof an antibody responsible for binding to antigen is the variableregion, the V-region. This region consists of two separate polypeptides.One polypeptide comes from the light chain and the other from the heavychain. In the intact antibody the variable region polypeptide from thelight chain extends into the constant domain of the light chain, thusgiving rise to an approximately 200 amino acid residues long polypeptidewith a total of two domains. In the intact antibody the variable regionfrom the heavy chain extends into the constant domains 1 to 3 givingrise to a 400 amino acid residues long polypeptide with a total of fourdomains. In the intact antibody two heavy chains combine with two lightchains. Thus an intact antibody contains two V-regions and is able tobind to two antigens at the same time. The constant regions of the heavychain interacts with the effector functions of the immune system.

FIG. 15 shows the effects of inoculations of normal human monocytes,polyclonal anti-RESA antibodies and polyclonal anti-MSP3 antibodies fromimmune human donors on parasitemia in the P.f.-HuRBC-BXN mouse model.

FIG. 16 compares the effects of chloroquine and polyclonal anti-MSP3antibodies from immune human donors on parasitemia in P.f.-HuRBC-BXNmice.

FIG. 17 shows the effects of inoculations of normal human monocytes,polyclonal anti-RESA antibodies and polyclonal anti-MSP3 antibodies fromimmune human donors on parasitemia in the P.f.-HuRBC-BXN mouse model.

FIG. 18 compares the effects of total IgG from immune donors andantibodies affinity purified on the MSP3b peptide on parasitemia inP.f.-HuRBC-BXN mice, Each curve represents the mean of results from sixmice.

FIG. 19 shows the effects on parasitemia in one P.f.-HuRBC-BXN mouse ofinoculations of human monocytes alone, in combination with an IgG1control antibody, and in combination with RAM1 IgG1.

Table I shows the results of screening of single clones from the threeseries after the fourth panning.

DETAILED DESCRIPTION OF THE INVENTION Example 1 Library Construction

Sampling of Peripheral Blood Lymphocytes

A volume of 100 ml peripheral blood was obtained after informed consentfrom each of 13 adults living in a malaria endemic area of Senegal, WestAfrica. The blood was collected in the anticoagulant ACD(adenine-citrate-dextrose) that conserves RNA better than heparin. Thesamples were transported to the laboratory where a brief centrifugationwas carried out, the buffy coat aspirated and cells suspended into anRNAse protecting buffer containing guanidinium HCl ,beta-mercaptoethanol and sarcosine. The material was then frozen inliquid nitrogen and shipped for further processing.

RNA Purification

RNA was isolated from the samples by acid phenol extraction according tothe procedure of Chirgwin et al. (Chirgwin et al. 1979). Messenger RNAwas converted to cDNA as described by Ørum et al. (1993).

PCR of Antibody Coding Genes

Amplification of antibody genes was carried out as described previously(Dziegiel et al. 1995). Briefly, separate reactions were performed forthe V_(H) genes, lambda light chain genes, and for the kappa light chaingenes. A pool of 12 individual primers was used for priming in the 5′region of the V_(H) genes (backward primers) and a pool of 3′ primerswas used for priming in the J_(H) region (forward primers). The detailsof the primers are given in Dziegiel (1995).

The amplification product of approximately 350 bp was purified from alow-melting point agarose by digestion of the agarose with agarase(Boehringer-Mannheim, Germany). This fragment was used as a template fora secondary extension PCR with primers complementary to the ends of theprimary product and additionally containing sequences corresponding tothe restriction enzymes NheI (5′) and ApaI (3′). Extended V_(H)fragments thus have a 5′ NheI site and a 3′ ApaI site. A pool of 8primers was used for priming in the 5′ region of kappa light chain genesand one primer for priming in the 3′ region. For amplification of lambdalight chain genes a pool of 12 backward primers and one forward primerwere used. The resulting fragment of approximately 700 bp was purifiedas above and used as template for a secondary extension PCR introducingthe restriction enzyme sites SfiI (5′) and AscI (3′). Extended kappachain genes thus have a 5′ SfiI site and a 3′ AscI site.

Polymerase Chain Reaction

Antibody genes were amplified from cDNA by PCR with separate reactionsfor V_(H) region genes, λ chain genes, and κ chain genes, respectively.PCR was performed in 100 μl volumes containing 0.2 mM dNTP's andreaction buffer supplied by the manufacturer (HT Biotechnology, UK), anequimolar mixture of primers totalling 20 μM (i.e. 0.65 μM of each V_(H)primer, 0.74 μM of each V_(κ)primer, 0.83 μM of each V_(λ)primer, 20 μMof the κ chain constant domain primer, 5 μM of each of the λ chainconstant domain primer, and 5 μM of each of the J-region primers), cDNA,and enzyme. An initial one min period at 94° C. was followed by additionof enzyme, and the cycle (94° C. for one min, 55° C. for one min, 72° C.for one min) was then repeated 30 times. Reactions were performed asdescribed (Ørum et al., 1993), except that a HYBAID Omnigenethermocycler was used.

Plasmid pFAB73HHui

The pFAB73HHui vector was developed from pFAB73H (Dziegiel et al., 1995;Engberg et al., 1996). The vector harbors an intact lacI^(q) geneencoding a highly efficient mutant of the lacI repressor. This ensures ahigh level of gene repression independent of the bacterial background.The sequences between the NheI-ApaI sites, and between the SfiI-AscIsites have been exchanged with sequences originating from a murineantibody against human insulin. This improves the efficiency of thesecond digestion and facilitates isolation of double digested plasmidduring the subsequent low-melting point agarose electrophoresis. Otherfeatures of the plasmid are identical to the pFAB4H. Briefly, pFAB73HHUIcontains the DNA fragment encoding amino acid residues 118 to 230 of thehuman γl H chain corresponding to constant domain 1 (C_(H)1). The C_(H)1gene extends in frame into a truncated version of gene III (ΔgIII) via aDNA fragment encoding a spacer and a trypsin cleavage site. ΔgIIIencodes a truncated version of surface protein pIII (ΔpIII) of thefilamentous phage, f1 (Bass et al., 1990; Ørum et al., 1993). The lacZpromoter and the 5′ part of the PelB leader gene were positioned infront of cloning sites for L chain genes. A DNA fragment containing aribosome binding site (RBS) and the 5′ part of the gene encoding thePelB leader was positioned 5′ to cloning sites for H chain variableregion (V_(H)) genes.

pFAB73HHUI permits insertion of V_(H) genes and L chain genes by atwo-step cloning procedure, first inserting V_(H) genes into restrictionenzyme sites NheI and ApaI, then inserting L chain genes into the sitesSfiI and AscI. All necessary genetic elements and the C_(H)1 are presentin the vector, thus minimizing the total number of PCR cycles.

Cloning of Antibody Genes

Amplification products from the secondary extension PCRs were separatedon low-melting point agarose followed by digestion with agarase. Theextended V_(H) fragments were digested with restriction enzymes NheI andApaI in two sequential reactions. The NheI-ApaI-digested V_(H) genefragment was purified on a low-melting point agarose gel followed byagarase digestion. The cloning vector, pFAB73HHUI, was digested withrestriction enzymes NheI and ApaI in two sequential reactions. TheNheI-ApaI-digested 5.4 kb plasmid was purified on a low-melting pointagarose gel followed by agarase digestion. The V_(H) gene was ligatedinto the plasmid, the ligation mixture was extracted with Phenol andChloroform, ethanol precipitated and electroporated into E. coliTop10Tet using an E. coli pulser set (BioRad). Bacteria were resuspendedin SOC medium (Sambrook et al. 1989), incubated at 37° C. for 1 h withshaking and then plated on LB agar (Sambrook et al. 1989) containing 50mg/l carbenicillin, 12.5 mg/l tetracycline and 2% glucose. Plates wereincubated overnight at 37° C. Colonies were washed off the plates withLB medium with carbenicillin, tetracycline and glucose and used to starta liquid culture, which at OD₆₀₀=1 was used to prepare the recombinantvector using Qiagen columns (Qiagen GmbH, Hilden, Germany) according tothe procedures recommended by the manufacturer. The purified vectorcontaining H chain genes was used as cloning vector for the cloning ofkappa and lambda light chain genes.

The extended kappa chain products were digested with restriction enzymesAscI and SfiI in two separate reactions. The kappa chain genes weresubsequently cloned into SfiI- and AscI-digested pFAB73HHUI harbouringV_(H) region genes.

Library

The resultant library contained 5×10⁷ different heavy chains, 1×10⁸kappa light chains and 1×10⁸ lambda light chains. Investigation of V_(H)genes from 66 clones by PCR and BstNI digestion demonstrated lack ofidentical digestion patterns thus suggesting acceptable diversity.

In this system, the heavy (H) chain fragment, Fd, is produced as afusion protein with a truncated version of the filamentous f1 phageprotein III (ΔpIII) (Bass ET al. 1990). H chain fusion proteinsassociate with intact light (L) chains in the periplasmic space to formFab heterodimers, which are fused to the ΔpIII protein. Upon infectionwith helper phage, the fusion proteins are positioned on one end of thephage together with a few copies of helper phage-derived wild-type pIII(Clackson et al. 1991; Hoogenboom et al. 1991). Thus, the Fab is able tobind to its specific antigen, and at the same time remains physicallyassociated with the phagemid DNA which harbors the genes encoding thatFab, i.e. a physical linkage has been established between recognitionand replication. This allows selection of a given antibody specificityand its corresponding gene from a large number of different Fab-phage(Marks et al. 1991).

Example 2 Selection

Production of Primary Phage Stock

A 50 ml culture of the library in LB medium with 50 mg/l carbenicillin,12,5 mg/l tetracyclin and 2% glucose was superinfected with VCSM13helper phage (Stratagene) at an OD₆₀₀=0.8. A multiplicity of infectionof 100 was used and the mixture was incubated at 37° C. with gentleshaking (50 rpm) for 1 h. Then the culture was diluted into 950 mlmedium as above without glucose and incubated at 30° C. overnight. Aftera 15-min spin at 10,000×g the supernatant containing phage wasprecipitated with PEG₆₀₀₀ and sodium chloride at final concentrations of4% and 0.5 M respectively. The supernatant was incubated for 1 h on iceand centrifuged for 30 min at 12,000×g. Precipitated phage wasresuspended in phosphate buffered saline (PBS) with 0.1% bovine serumalbumin (BSA) and used immediately. The total number of colony formingunits was 1.3×10³ (determined as per Sambrook et al. 1989).

Selection of Specific Binders in the First Panning

Biotinylated MSP-3₁₉₄₋₂₅₇ was coupled to Dynabeads M-280 Streptavidin(cat no. 112.05) as described by the manufacturer. After incubating theantigen-coated beads with phage in an end-over-end mixer for 1 h, beadswere captured with a magnetic particle concentrator (MPC-6, Dynal Catno. 120.02) for 5 min. and then washed 6 times in 10 ml PBS with 0.05%Tween20 for 2 min. After the last wash beads were resuspended in 1 mlPBS with 1 mg trypsin (Worthington, USA) and phage was eluted during aone-hour incubation at 37° C. Then a volume of 3 ml of exponentiallygrowing Top10 with OD₆₀₀=1 was added and the mixture was incubated at37° C. for 30 minutes to allow eluted phage to attach to E. coli. Thebacteria were finally plated on LB agar (Sambrook et al. 1989)containing 50 mg/l carbenicillin, 12.5 mg/l tetracyclin and 2% glucoseand incubated overnight at 37° C. Colonies were washed off the plateswith LB medium and stored as a glycerol stock at −80° C. or grown forproduction of phage and DNA. DNA was produced as described.

Production of Phage for subsequent Pannings

Phage was produced by super-infecting a 15 ml culture of the glycerolstock at OD₆₀₀=0,6 (after approx. 3 hours) by addition of helper phageVCSM13 (Stratagene, USA). Multiplicity of infection was approx. 100.After 1 hour of gentle shaking the culture was diluted into 200 ml ofLB-medium and supplemented with isopropyl-β-D-thiogalactopyranoside(IPTG) to give a final concentration of 50 μM, 70 mg/l kanamycin, 50mg/l Carbenicilin and 12,5 mg/l tetracycline The culture was grown overnight. Phage was isolated as above except that it was cleared beforepanning by two additional spins of each 20 minutes at 17,320×g.

Selection of specific Binders in Subsequent Pannings

Subsequent to the first panning, three series of panning were carriedout in parallel, to a total of 4 rounds. In the first series, the numberof beads was reduced by a factor of 5 for each subsequent panning. Inthe second and the third series the number of beads was reduced by afactor of 10 and 20, respectively, from one panning to the next. Afterthe fourth panning single colonies from all three series were grown insterile microtiter plates for production of Fab-ΔpIII.

Testing of Final Eluate

The eluate from the fourth panning was screened for the presence ofFab-ΔpIII producers and antigen binders, respectively. Fab-ΔpIII wasproduced by growing individual colonies in microtiter wells. A total of376 clones were tested. Supernatant from 23 clones contained freeFab-ΔpIII and 17 of these were able to bind to MSP-3₁₉₄₋₂₅₇ (see tableI). The antibody genes of the 17 clones were sequenced using Big Dyeaccording to the procedures of the manufacturer (Applied Biosystems,USA).

The selection procedure resulted in isolation of three distinct clonesdesignated RAM1, RAM2 and RAM3.

Example 3 Determination of Specificity

The reactivity of the clones with native malaria antigen wasdemonstrated.

Direct ELISA

ELISA plates (Maxisorb, NUNC 4-39454, Denmark) were coated with 10 to650 ng per well of purified recombinant MSP-3₂₂₋₂₅₇ or MSP-3₁₉₄₋₂₅₇ orof the peptides MSP-3₁₉₀₋₂₁₇ or MSP-3₂₁₁₋₂₃₇ in PBS and used forstandard ELISA with undiluted supernatant or supernatant diluted inPBS-BSA as previously described (Dziegiel et al.1991; Dziegiel et al.1995). After wash, goat anti-human Fab (Sigma A8542) or anti-human IgGFc (Sigma A9544) antibodies conjugated to alkaline phosphatase wasapplied as detection antibody. Finally, p-nitrophenyl phosphate (Sigmaphosphatase substrate tablets, 104-105) was used as substrate. Colordevelopment was measured as OD₄₀₅-OD₄₉₀, see FIG. 1. RAM1 and RAM2 bothbind to the short MSP-3₁₉₄₋₂₅₇ and the long MSP-3₂₂₋₂₅₇ recombinantversions of the MSP-3 antigen. In contrast, RAM3 binds only the shortMSP-3₁₉₄₋₂₅₇ version. The controls (anti-Hib CP and PBS 1% BSA) do notreact with the antigen.

Antigen Competition

To wells containing 50 μl of a fixed dilution of Fab-ΔpIII were added 50μl of various dilutions of competition antigen, MSP-3₁₉₃₋₂₅₆. Coating ofwells with MSP-3₁₉₄₋₂₅₇ and detection of bound antibody were carried outas above. The concentration of competition antigen ranged from 4 nM to1250 nM. Each dilution of competition antigen was tested in duplicate,see FIG. 2. All three clones are competed by soluble recombinantMSP-3₁₉₄₋₂₅₇ antigen. This demonstrates that the antibodies are directedto the conformation of the antigen in solution and not to somedenaturation-dependent conformation e.g. a plastic-binding dependentepitope.

Immunofluorescence Microscopy

Parasitized erythrocytes from in vitro culture of P. falciparum clone3D7 were attached to a slide, air dried and then fixed with acetone for5 minutes at room temperature as described (Druilhe et al. 1987).Fab-ΔpIII or intact IgG1 was added and after 30 minutes slides werewashed in PBS. Bound Fab-ΔpIII was detected with FITC-conjugated goatanti-human Fab (Sigma F5512) diluted 1:25. Hib-CP specific antibody wasused as control in corresponding concentrations. To stain theDNA-containing infected erythrocytes propidium iodide was added to thesecondary antibody solution. The late schizont stage was intensivelystained with propidium iodide due to its high content of merozoite DNA.The RAM1 Fab stained exactly this stage. In contrast, red cellscontaining small amounts of parasite DNA did not bind to the RAM1 Fab.Red cells with small amounts of DNA represents early stages, i.e. ringstage and trophoblasts, see FIG. 8.

Immunoblotting

Purified parasites were solubilized in 2% Triton X-100 by treatment withultrasound 4×15 seconds on ice and incubation for 2 hours on ice beforebeing mixed with sample buffer, heated to 100° C. for 10 minutes andsubjected to SDS-PAGE in a MOPS-buffered 4-12% gradient gel (NOVEX)-Proteins were transferred electrophoretically to a PVDF membrane(Immobilon, Millipore) by wet blotting, blocked by drying the membraneand by incubation in 0,1% Tween20 and 0,2% I-Block (Tropix Cat. No.AI300) immediately before use. The blot was incubated for 3 hours withanti-malaria antibody RAM1, RAM2 or RAM3 produced as IgG1, washed 3×5minutes with PBS supplemented with 0.1% Tween20 and 0,2% I-Block, andthen incubated for 1 hour with alkaline phosphatase conjugated protein G(Pierce Cat no.32391). The membrane was washed 3 times as above.Finally, CSPD chemiluminescent substrate (Tropix Cat. no.CD100R) wasadded to the blot and light emission was detected with the AlphaInnotech FluorChem 8000 system. Hib-CP specific antibody was used asnegative control in corresponding concentrations, see FIG. 7. RAM1reacted with parasite proteins of relative molecular weights ofapproximately 51 and 53 kD, respectively. RAM2 reacted with parasiteproteins of relative molecular weights of approximately 14 and 64 kD,respectively. RAM3 reacted with a parasite protein of approximately64kD. Molecular weights in the same range have been observed withpolyclonal antibodies from man and rabbit (Oeuvray et al, 1994; McCollet al, 1994). The background for the variation in molecular weights ofthe bands recognised by these two antibodies could be post-translationalmodification, proteolytic processing or the presence of subpopulationsof molecules with distinct conformations. The latter possibility istempting due to the alleged propensity of MSP-3 to obtain intra- orintermolecular coiled-coil conformations (McColl et al, 1994) laterextended by Trucco et al (2001) to pertain to other merozoite specificproteins like MSP-1 and MSP-6. Such tertiary conformation will influencethe electrophoretic mobility and could thus be responsible for theobserved range of molecular weights. The band of 14 kD is believed to bedue to proteolytic degradation. There have been no other reports ofbands in this range.

Flow Cytometry

Purified schizonts were permeabilized and fixed by incubation in 32%ethanol for 30 minutes on ice. Parasites were then washed in PBS with 1%BSA, incubated over night with antibody (Fab-ΔpIII or IgG1), washedtwice with PBS supplemented with 1% BSA and incubated for 30 minuteswith FITC-conjugated goat anti-human Fab (Sigma F5512) diluted 1:25.Cells were analyzed in a Coulter EPICS-2 flow cytometer. Hib-CP specificantibody was used as control in corresponding concentrations. Fixedinfected and fixed non-infected red cells were stained with propidiumiodide and compared to enable gating on infected red cells, see FIG. 4.

Epitope Mapping

Our mapping of the epitopes relates to a previously used arbitrarydivision of MSP-3₁₉₄₋₂₅₇ into three peptides (Oeuvray et al. 1994)termed MSP-3a, MSP-3b and MSP-3c (Oeuvray et al. 1994). The biologicalrelevance of the dissection of MSP-3₁₉₄₋₂₅₇ is that polyclonalantibodies directed to MSP-3b, corresponding to MSP-3₂₁₁₋237 show aclear anti-parasitic effect in ADCI and in the mouse model (Badell etal. 2000).

Two peptides, MSP-3₁₉₀₋₂₁₇ and MSP-3₂₁₁₋₂₃₇, were synthesized and usedin ELISA. RAM1 reacts predominantly with MSP-3₂₁₁₋₂₃₇. RAM2 reacts tothe same extent with MSP-3₁₉₀₋₂₁₇ and MSP-3₂₁₁₋₂₃₇, and RAM3 reactedonly weakly with these two peptides—see FIG. 9.

Additionally, the entire sequence MSP-3₁₉₄₋₂₅₇ was covered by synthesisof 34 sets of two peptides comprising contiguous amino acid sequences.The two peptides were linked by a proprietary linker substituting twoamino acid residues on the location of the linker (PEPSCAN). Thus onesingle set of peptides covers a stretch of 31 amino acid residues withtwo amino acid residues replaced in positions 15 and 16 by theproprietary linker residues. The linked peptides were furthermorechemically coupled to the matrix to allow washing and repeated use. Theoverlap between two adjacent sets of peptides was two amino acidresidues. This collection of peptides was examined for reactivity withRAM1 and RAM2 produced as IgG1. The result is presented in FIG. 10. RAM2reacts with the amino-terminal, the middle and the carboxy-terminal partof the antigen but yields the highest reactivity with the middle part.RAM1 reacts only weakly with the peptides produced by this method. RAM3was tested as Fab-ΔpIII in PEPSCAN without response.

The interpretation of the results is that RAM1 as well as RAM2 reactwith conformational, non-linear epitopes comprising amino acids residuesbeing non-contiguously positioned in the antigen.

The epitope for RAM1 certainly comprises residues in the middle part.The contribution of residues in the amino-terminal and thecarboxy-terminal part of the antigen is uncertain. The amino-terminalpart of the antigen represented by the peptide MSP-3₁₉₀₋₂₁₇ comprisesonly heptad repeat sequence. The first residues of MSP-3₂₁₁₋₂₃₇ are partof a heptad repeat making it likely that the epitope for RAM1 is thec-terminal part of MSP-3₂₁₁₋₂₃₇. The region represented by residues220-230, ILGWEFGGGVP (SEQ ID NO: 10), is outstanding by being present inMSP-3 as well as the other Plasmodium falciparum antigen MSP-6 (Truccoet al. 2001).

Based on the PEPSCAN results we conclude that RAM2 reacts with residuesfrom all parts of the antigen but the major contribution is from themiddle part.

Antibody Competition

The studies of binding to truncated antigens were supplemented with anantibody competition experiment. Fab fragments from clone RAM3 wereproduced in two versions. One version was fusion protein tagged with atruncated phage protein, ΔpIII, designated Fab-ΔpIII, and anotherversion was normal Fab fragments without the ΔpIII tag. The ΔpIII-tag isreadily detected using an antibody directed to the phage pIII protein.Thus Fab without ΔpIII can be present as competitor but only Fab-ΔpIIIwill be detected.

Amounts of RAM1, RAM2 and RAM3 Fab-ΔpIII yielding OD₄₀₅ of approximately1 in ELISA on a coating of MSP-3₁₉₄₋₂₅₇ were used. The binding of theFab-ΔpIII was then competed by the addition of increasing amounts ofcompetitor, RAM3 without ΔpIII. FIG. 13 shows that Fab RAM3 is able tocompete Fab-ΔpIII RAM1 to the same extent as it competes Fab-ΔpIII RAM2.This demonstrates that binding of the clones RAM1 and RAM2 is dependenton the epitope used by clone RAM3. The lower competitive effect of RAM3on the binding of Fab-ΔpIII RAM3 could be explained by a higher affinityof RAM3 as compared to RAM1 and RAM2. The effect of this would be thatthe competitive effect of RAM3 towards RAM1 and RAM2 would be largerthan towards RAM3 itself, see FIG. 13.

RAM3 was only reactive with the intact panning antigen and did not reactwith truncated versions. Furthermore RAM3 did not react with parasitesin flow cytometry. However, the antibody competition experiment aboveshows that the epitopes for RAM1, RAM2 and RAM3 are closely dependent beit by direct steric hindrance due to overlap of epitopes or an effect ofbinding of RAM3 on the correct folding of the antigen.

We then tested if a malaria immune serum obtained from an individualliving in a malaria-endemic area contains antibodies that will interferewith the binding of clone RAM1to MSP-3₁₉₄₋₂₅₇. Clone RAM1 was producedas IgG1 and conjugated to FITC. The FITC-IgG1 was used in an assayidentical to ELISA except for the detection of binding that was carriedout by measurement of fluorescence in a microtiter plate basedfluorometer. The amount of FITC-IgG1 was adjusted to give maximum effectof competition. Increasing amounts of competitor immune serum was addedto individual well of the microtiter plate. A serum from a non-immuneEuropean donor was used as negative control. The malaria immune serumcontained antibodies that significantly inhibited the binding of RAM1IgG1. For technical details see below.

Method: Immune Serum Competition

MSP-3₁₉₄₋₂₅₇ coated ELISA plates (Maxisorb, NUNC 475515, Denmark) wasincubated with serial dilutions of either immune serum (from a malariaimmune individual) or control serum. After two hours the plate waswashed 5 times with PBS 0,05% Tween 20. Then FITC-conjugated RAM1 IgG1antibody was applied in a dilution yielding maximum reactivity.FITC-conjugation was performed as described by the manufacturer(Molecular Probes, cat. No. F-6434). The plate was incubated for onehour, then it was washed 5 times and subsequently relative fluorescencewas determined using a Polarstar fluorescence reader (BMG, Germany).

We conclude that the epitope recognized by RAM1 on the antigen is indeedone that is also recognized by the immune system of a malaria immuneindividual. This substantiates the clinical in vivo relevance of theRAM1 clone and thus also of clones RAM2 and RAM3 as the three clonesbind to very closely related epitopes, see FIG. 13.

Example 4 Production of Eukaryotic IgG1 and IgG3

Methods

Plasmids

The eukaryotic expression of intact antibodies was performed with thetwo plasmids, pLNOH2 and pLNOK (Norderhaug et al. 1997).

The structure of these plasmids and their relation to the assembledantibody is shown in FIG. 12 and FIG. 14.

Briefly, the V-regions of the plasmids are exchanged by cutting withBsmI in the 5′ end and by cutting with HpaI or BsiWI or HindIII in the3′ end. Before ligation of vector and V region, the V regions of RAM1,RAM2 or RAM3 need to be cut with the same restriction enzymes as thevectors. VH regions are then ligated to pLNOH2 and VK regions areligated to pLNOK. The constant region of pLNOH2 can be exchanged bycutting with BamHI and HindIII, isolating the vector part andintroducing (by ligation) another constant region cut with the sameenzymes.

pLNOH2 is a vector for expression of heavy chain V-region genes incombination with any constant fragment (Fc) gene. Two versions of thevector were used, one with the human γl constant fragment gene (allotypeG1m(a, z)) and another with the human γ3 gene (allotype G3m(b)), toenable recombinant expression of the two isotypic variants of theantibody.

pLNOK is a vector for expression of kappa light chain V-region genes incombination with the kappa constant domain gene. The common features ofthe two vectors are briefly described below. The CMV promoter issituated 5′ to a gene casette containing the leader region of murineimmunoglobulin genes, the V-region gene inserted between restrictionsites BsmI and BsiWI followed by an intron and the entire genomicC-region with a poly-A signal in the 3′ end. The intron comprises spliceacceptor and splice donor signals for correct processing of the mRNA.Non-abundant restriction sites flank the V-regions genes and areintroduced by PCR into the gene fragments to be cloned. Additionalfeatures of the vectors are, ampicillin resistance marker for selectionin E. coli, f1-origin of replication, Neomycin resistance selectionmarker for stable expression and the SV40 origin of replication to allowtransient expression.

In order to produce a complete antibody, the plasmids (both pLNOH2 andpLNOK) are cotransfected into CHO cells. The V and C regions areassembled by mRNA splicing. When co-transfected in one cell, e.g. a CHOcell, the cell produces complete and correctly assembled antibodies.

As an alternative, the genes can be assembled into one vector which thusharbours the VH, CH , VL and CL domains.

PCR of Antibody Genes

Two separate PCR amplifications were performed for each antibody, onefor the heavy chain and one for the light chain. The templates werepFAB73H+RAM1, pFAB73H+RAM2 and pFAB73H+RAM3. For amplification of theRAM1 heavy chain V-region fragment one primer designated EuHVH-1 wasbased on the 5′ sequence of RAM1 V_(H) (indicated in bold) and furthercontained the recognition sequence of restriction enzyme BsmI (indicatedby underlining and italics):

EuHVH-1: (SEQ ID NO: 79) 5′ ggt g tg cat tc c cag gt n caa ttg gt r ca rtc y g 3′

Ambiguous bases (highlighted by underlining) were introduced to makethis primer compatible with the primer set published by Dziegiel et al.1995.

The other primer, EUHJH, was complementary to the 3′ sequence of RAM1V_(H) (indicated in bold) and contained the additional sequence of therestriction enzymes HindIII, BsiWI and HpaI (indicated by underlining)and the complementary sequence to the splice donor sequence 5′ AGGTGAGT3′ (indicated in capital letters):

EuHJH: (SEQ ID NO: 80) 5′ gt cca agc ttc gta cgt taA CTC ACC Tga rga gacrgt gac c 3′          HindIII    BsiWI     HpaI

For amplification of the RAM1 light chain V-region gene fragment twoprimers designated EuHVKuniversal and EuHJK-14a were used. EUHVKuniversal was made compatible with the primer set published by Dziegielet al. 1995 by taking advantage of the invariant 5′ sequence of allkappa chain genes derived from this primer set (indicated by doubleunderlining).

The primer further contained the recognition sequence of restrictionenzyme BsmI (indicated by bold) and part of the pelB leader sequencefound in the phage display vector (indicated by underlining):

EuHVKuniversal: (SEQ ID NO: 81) 5′ ttg tta tta ctc gcg gcc cag ccg g tgcat tcc gac atc 3′     |---------- PelB leader seq. -----||BsmI    |  |-> Vκ

EuHJK-14a was based on the same principle as EuHJH shown above,complementarity to the 3′ sequence of RAM1 V_(K) (indicated in bold),the sequence of the restriction enzymes HindIII, BsiWI and HpaI(indicated by underlining) and the sequence complementary to the splicedonor sequence 5′ A CTC ACG T 3′ (indicated in capital letters):

EuHJK-14a: (SEQ ID NO: 82) g tcc aag ctt cgt acg tta acA CTC ACG Ttt gatctc cag cct gg

For amplification of the RAM2 heavy chain V-region fragment one primerdesignated EuHVH-2 was based on the 5′ sequence of RAM2 V_(H) (indicatedin bold) and further contained the recognition sequence of restrictionenzyme BsmI (indicated by underlining and italics):

EuHVH-2: (SEQ ID NO: 83) ggt gtg cat tc c cag gtg caa ttg cag gag tc s g

Ambiguous bases (highlighted by underlining) were introduced to makethis primer compatible with the primer set published by Dziegiel et al.1995.

The other primer, EuHJH is described above.

For amplification of the RAM2 light chain V-region gene fragment twoprimers designated EuHVKuniversal and EuHJK-15 were used. EuHVKuniversalis described above.

EuHJK-15 was based on the same principle as EuHJK-14a shown above,complementarity to the 3′ sequence of RAM2 V_(K) (indicated in bold),the sequence of the restriction enzymes HindIII, BsiWI and HpaI(indicated by underlining) and the sequence complementary to the splicedonor sequence 5′ A CTC ACG T 3′ (indicated in capital letters):

EuHJK-15: (SEQ ID NO: 84) g tcc aag ctt cgt acg tta acA CTC ACG Ttt gatctc cac cc

For amplification of the RAM3 heavy chain V-region fragment the sameprimers were used as for RAM1 (see above)

For amplification of the RAM3 light chain V-region gene fragment twoprimers designated EuHVKuniversal and EuHJK-8 were used. EuHVKuniversalis described above.

EuHJK-8 was based on the same principle as EuHJK-14a shown above,complementarity to the 3′ sequence of RAM3V_(K) (indicated in bold), thesequence of the restriction enzymes HindIII, BsiWI and HpaI (indicatedby underlining) and the sequence complementary to the splice donorsequence 5′ A CTC ACG T 3′ (indicated in capital letters)

EuHJK-8: (SEQ ID NO: 85) gt cca agc ttc gta cgt taa ctt ctA CTC ACG Tttgat ytc cac ctt gg

The PCR were performed using Gene Amp PCR System 9600, AmpliTaq Gold andthe supplied buffers. Primers were used in a final concentration of 0,2μM. The PCR cycling included the following steps: 10 min. at 95° C. (hotstart, activation of AmpliTaq), 14 cycles of 94° C. for 30 seconds, 40°C. for 30 seconds and 72° C. for 30 seconds and finally one cycle at 72°C. for 10 min.

Amplification products of the expected sizes were isolated by gelelectrophoresis, purified with Gene Clean (Bio 101, Inc., Calif, USA)and ligated into pGem-T Easy (Promega, USA) according to themanufacturer.

Ligations were used to transform electrocompetent XL-1 blue(Stratagene). Subsequently the cells were plated on LB containing X-galand IPTG as described in Sambrook et al. 1989. White clones weresequenced using Big dye (Applied Biosystems, USA) and the primers SP-6and T7 as recommended by the manufacturer. The sequence reactions wereapplied to ABI PRISM Sequencer 310 (Perkin 5 Elmer, USA).

For each heavy and light chain a clone was selected that was eitheridentical to the template or that did not alter the deduced amino acidsequence.

The selected clones were digested with BsmI and BsiWI or with BsmI andHindIII. The fragments were then ligated into the appropriate vectordigested with the same enzymes.

Cell Line

CHO cells were used as eukaryotic host cells for stable expression ofintact antibodies.

Transfection and Expression

Cells were grown to 50-80% confluency in Ham's F-12 Medium supplementedwith 10% inactivated Foetal Bovine Serum (Life Technologies). Cells werethen transfected with Lipofectamine (Life Technologies) according to theprocedures of the manufacturers. The following day transfected cellswere transferred to microtiter plates to select clones by limitingdilution. After approximately 14 days wells containing single cellcolonies were tested for production of anti-MSP-3 antibodies in ELISA.Cells from approx. 20 wells giving the highest response in the ELISAassay were transferred to new wells in a 24 well microtiter plate, grownto confluence, transferred to new wells in a 6 well microtiter plate,grown to confluence and finally transferred to two 25 cm² TC flasks.Cells in one flask were grown to confluence and sebsequently frozen.Cells in the second flask were grown for approx. 4 weeks whereupon thesupernatant was tested for production of anti-MSP-3 antibodies in ELISA.The clone giving the highest response in the ELISA assay was used for asecond round of limited dilution. The clone from the second round oflimited dilution giving the highest response in the ELISA assay wasgrown to an appropriate number of cell to be used for 1) freezing and 2)expression of antibody. For expression cells were plated to approx. 10%confluence in triple TC flasks (NUNC, Denmark) and grown for approx. 4weeks. The supernatant was centrifuged 20,000×g for 30 minutes beforeuse in subsequent assays to remove cellular debris.

Immunochemistry

The composition of human antibodies in the supernatant from the cellculture was examined by capture ELISA. Human antibodies were captured ona coating of goat anti-human Fab fragments (Sigma I5260) and detectedwith mouse anti-human IgG1(Zymed 05-3600) or IgG3 (Zymed 05-3300),respectively, followed by rabbit anti-mouse conjugated to horse radishperoxidase (DAKO P260). ELISA for reactivity with specific antigen wasperformed as described under Example 3. Furthermore, the IgG1version ofRAM1, RAM2 and RAM3 were used for Western blotting and RAM1 IgG1 wasused for flow cytometry.

Capture ELISA for Quantitation of IgG

Capturing of recombinant IgG was done with goat anti-human IgG specificfor the Fc-portion 1:5,000 (Sigma I2136), detection was performed withalkaline phosphatase conjugated goat anti-human kappa chain 1:10,000(Sigma A3813). Purified human recombinant anti-D IgG1, kappa (751 pg/mL)and IgG3, kappa (135 μg/mL), respectively, were used as standards.Standard IgG had identical allotypes as malaria antibodies. ELISAprocedures and buffers were as described in Dziegiel et al. 1991.

Results

The V_(H) and V_(K) genes derived from the anti-MSP-3 antibodies werecombined with constant domain genes in plasmids pLNOH2 and pLNOK,respectively. Two different versions of the heavy chain vector, pLNOH2,were used, one version containing the γ1 constant domain gene and theother containing the γ3 constant domain gene. These two vectors wereused for production of an IgG1 version as well as an IgG3 version of theanti-MSP-3 antibodies. The composition of the antibodies wasinvestigated by immunochemistry in ELISA. The specificity of the IgG1and IgG3 versions was tested with ELISA technique.

Example 5 Purification of Eukaryotic IgG1 and IgG3

After ammonium sulfate precipitation (70%) of cleared culturesupernatant, pelleted proteins were resuspended in water. Bufferexchange to 70 mM Sodium acetate pH 5.0 was performed by dialysis.Recombinant IgG was purified using DEAE Sepharose FF (Pharmacia BioTech)followed by applying the flow through to an ABx column (Baker Bond). Thebuffer was changed to 50 mM Bicine pH 8.5 and the IgG eluted with agradient of 50 mM Bicine supplemented with 0.5 M NaCl pH 8.5. Ifnecessary antibody containing fractions from the Abx column areprecipitated by ammonium sulphate as above and applied to the gelfiltration column (Superdex200). The purity was checked by gelchromatography using Superdex200 and by SDS-PAGE followed by silverstaining.

Example 6 Characterization of the Biological Activity of RecombinantAntibodies by ADCI and Other Functional and Parasitological Tests

These assays reflect the clinical status of protection. The ADCIreflects the capacity of individual IgG to inhibit P. falciparum growthin vitro by cooperation with human blood monocytes as described inBouharoun-Tayoun et al. (1995). The P. falciparum merozoite phagocytosisassay in the presence of antibody has been checked among the 200inhabitants from Dielmo where it closely reflects the clinical status ofthe inhabitants.

ADCI and phagocytosis assays may be performed with the selectedantibodies and human monocytes following standardized procedures.

For antibodies expressed with Fc portions in eukaryotic cells, theclassical ADCI assay may be used, as described by Shi et al. 1999.

The parasites are cultured in the presence of recombinant antibodies andhealthy donor derived monocytes. In order to determine the specificgrowth inhibition (SGI) of ADCI described below, the appropriatecontrols are included.

${S\; G\; I\mspace{14mu}{of}\mspace{14mu} A\; D\; C\; I} = {100 \times \left( {1 - \frac{\left( \frac{\%\mspace{14mu}{mean}\mspace{14mu}{parasitemia}\mspace{14mu}{with}\mspace{14mu}{test}\mspace{14mu}{IgG}\mspace{14mu}{and}\mspace{14mu}{monocytes}}{\%\mspace{14mu}{mean}\mspace{14mu}{parasitemia}\mspace{14mu}{with}\mspace{14mu}{test}\mspace{14mu}{IgG}} \right)}{\left( \frac{\%\mspace{14mu}{mean}\mspace{14mu}{parasitemia}\mspace{14mu}{with}\mspace{14mu}{control}\mspace{14mu}{IgG}\mspace{14mu}{and}\mspace{14mu}{monocytes}}{\%\mspace{14mu}{mean}\mspace{14mu}{parasitemia}\mspace{14mu}{with}\mspace{14mu}{control}\mspace{14mu}{IgG}} \right)}} \right)}$

Monocytes are isolated by the lymfoprep method, and by adhesion toautologous plasma coated plastic tissue culture Petri dishes (Nunc).

The malaria parasite may be grown as described by Trager and Jensen 1976in 96 well tissue culture plates. The effect of IgG's and monocytes isdetermined in 48-hour cultures with an initial parasitemia of 0,5%. Fortests with monocytes these are activated by rhIFN-γ and added to eachwell.

After 48 hours the parasitemias are assessed by flow cytometry asdescribed by van der Heyde et al. (1995). The cultures are after 48hours incubated with hydroethidine which is a vital dye that isconverted to ethidium by metabolically active cells. Ethidium interactswith the parasite derived DNA in infected cells and thereby allowsdiscrimination between infected and non-infected erythrocytes byflowcytometry (Shi et al., 1999).

A flow cytometry based phagocytosis assay developed by Kumaratilake andFerrante (2000) may be used to determine the opsonization andphagocytosis mediating abilities of the produced antibodies. This assayexplores the ability of neutrophils to quench FITC-conjugatedmerozoites. This assay is adapted to detect phagocytosis by otherleukocytes (such as monocytes) as an effect of antibody opsonization(Kushmith & Druilhe, 1983.

Furthermore, the antibodies produced may be tested in vivo in the mousemodel developed by Badell et al (1995) & (2000). This model is based onthe possibility of obtaining a sustained P. falciparum growth inimmunocompromised mice reconstituted with human erythrocytes as well ashuman monocytes.

Studies of this nature are described below. The antimalarial biologicalactivity of RAM1 IgG1 and RAM1 IgG3 recombinant antibodies was assessedusing both in vitro and in vivo assays previously established asreflecting immune mechanisms of defense mediating protection against P.falciparum in human beings (Druilhe et al., 1995; Bouharoun-Tayoun etal., 1995; Badell et al., 1995).

In Vitro ADCI Studies

In vitro studies were aimed at measuring the monocyte-dependantantibody-mediated mechanism of cooperation named ADCI(Antibody-Dependant Cellular Inhibition of P. falciparum) previouslyfound to correlate closely with the status of protection obtained bypassive transfer of African human IgG in to P. falciparum infected Thaireceivers (Sabchareon et al., 1991; Bouharou-Tayoun et al., 1990), and amechanism that has been at the origin of the identification of theMerozoite Surface Protein-3 (MSP-3) molecule on the merozoite surfaceand the identification of the MSP-3. b epitope within the C-terminusregion of the antigen (Oeuvray et al., 1994). RAM1 IgG1and RAM1 IgG3were studied in the ADCI assay alongside with positive controls, namelythe adult African IgG pool which proved effective upon passive transferin P. falciparum infected subjects, an affinity-purified antibodyagainst the MSP-3. b peptide, and an anti-MSP-3 C-terminus antibodyobtained by immuno-purification of the same African IgG pool upon theC-terminus recombinant antigen expressed in the His-tail vector.Negative controls included total European IgG from individuals who havenot been travelling in malaria endemic areas, antibodies absorbed fromAfrican subjects upon the RESA (Ring Infected Surface Antigen) and theMerozoite Surface Protein-1 (MSP-1) antigen. The study was repeated 3times using 3 different normal blood monocytes donors and using eitherthe Ouganda Palo Alto strain or the 3D7 clone derived from the NF54strain of P. falciparum.

Positive Control IgG (PIAG) was purified from a serum pool obtained fromAfrican adults living permanently in a rural area of Ivory Coast wheremalaria is holoendemic. They were selected on clinical andepidemiological grounds (Sabchareon et al., 1991). They experiencednumerous malaria attacks in childhood and were free of symptoms andheavy parasitemia and thus regarded as immune individuals(Bouharoun-Tayoun et al., 1990). The IgG was extracted by ion-exchangechromatography on DEAE-sephadex (Pharmacia). The IgG containingfractions were pooled, protein content and antibody level weredetermined by Bicinchoninic acid protein determination reagents (Sigma)and IFA respectively. The protein content was found to be 16.2 mg/ml andIFA endpoint titre of the preparation was 1:52,000. Final concentrationof 2 mg/ml (10% of the serum concentration, 650 IFA titre) was used inADCI assay (Bouharoun-Tayoun et al., 1990).

Negative Control IgG ( NIG) was similarly prepared as above using acommercially available pool from more than 1,000 healthy French blooddonors (Biotransfusion CRTS, Lille, France). The IFA test was negativeat 1:200.

The indirect immunofluorescent antibody (IFA) test was performed asreported earlier (Bouharoun-Tayoun et al., 1990). Briefly, a thin filmof P. falciparum 3D7 schizont-infected RBCs was incubated with serialdilutions, starting from 1:200 of the recombinant antibodies or IgGs inPBS (pH 7.4) for 30 min at 37° C. inside humid chamber. Alexa fluorconjugated goat anti-human IgGs (Molecular Probe, USA) at 1:300 dilutionin PBS was used to detect the bound immunoglobulins. The endpoint titrewas the highest dilution of the antibodies which produced visiblespecific immunofluorescence.

Parasite culture: P. falciparum 3D7 clone from NF54 and PaloAlto werecultured in RPMI 1640 medium supplemented with hypoxanthine, 0.5%albumax (Gibco BRL), sodium bicarbonate, HEPES, penicillin andstreptomycin in AB⁺Rh⁻RBCs. Parasites were synchronized by alternatesorbital treatment and plasmagel floatation. On the day of ADCI assay,parasites were prepared from plasmagel floatation.

ADCI assay: Two P. falciparum strains, the 3D7 clone derived from NF54and the PaloAlto strain were used. ADCI assay was performed as described(Bouharoun-Tayoun et al., 1990) with some modifications. Normalmonocytes were obtained from healthy donors without exposure to malariainfection and separated from peripheral blood mononuclear cells byadherence on 96 wells flat-bottom culture plates (TPP, Switzerland).Donor's plasma was coated on the plate for better attachment.Mononuclear cells which will contain 2×10⁵ monocytes were distributedinto each well. Well synchronized P. falciparum schizont stage adjustedto 0.5% parasitemia with final haematocrit of 2% were added to eachwell. The anti-MSP3 RAM1 recombinant antibodies were added at threedifferent concentrations in respect to IFA titre of 200, 500 and 1000,to each well with and without monocytes. Final volume in each well wasadjusted to 100 μl. In addition to the control recombinant IgG1 andIgG3, the following control were run simultaneously in each plate: a)culture without monocytes, b) culture with monocytes, c) culture withNIG, d) culture with monocytes and NIG, e) culture with PIAG, f) culturewith monocytes and PIAG. 50 μl of RPMI containing 0.5% albumax,penicillin and streptomycin were added to each well at 48h and 72 h. Theassay lasted 96 h and at the end of the assay parasitemia was determinedby both microscopic counting of more than 50,000 RBCs on Giemsa stainedfilm and by FACS after staining with hydroethidine.

The specific growth inhibitory index (SGI) which takes intoconsideration the possible inhibition induced by monocytes or antibodiesby themselves, was calculated as described above.

Determination of parasitemia was undertaken as reported by van der Heydeet al. (1995). Parasite pellet from each ADCI well was incubated withfreshly diluted 200 μl of hydroethidine 50 μg/ml in PBS pH 7.4 for 20min at 37° C. in the dark. After incubation, the parasite pellets werewashed 2 times with PBS and resuspended in a final volume of 700 μl inthe fluorescence-activated cell sorter (FACS) tubes. Data acquisitionand analysis were performed on FACScalibur (Becton-Dickinson, San Jose,Calif.) . The detectors of forward and side scatter were set inlogarithmic mode and 100,000 cells were counted. Both infected anduninfected RBCs were gated in the analysis and the percentage ofparasitemia was determined by the use of Cellquest-Pro program(Becton-Dickinson). Parasitised red blood cells were distinguishablefrom monocytes on the basis of size and intensity of fluorescence.Uninfected human AB⁺ Rh⁻ similarly treated with hydroethidine wasassigned to region 1 and infected RBCs to region 2. The percentage ofmetabolizing or healthy parasites was similar to that obtained bymicroscopy.

TABLE II Specific Growth Inhibitory Index (SGI) of the recombinant humanRAM-l IgG1 and IgG3 subclasses against MSP3 antigen. Antibody μg/ml inADCI IFAT in ADCI SGI % DI % IgG1 anti-MSP3 40 50 72(±7) 4(±8) 20 1066(±3) 0(±0) Control IgG1 40 0  2(±3)  9(±16) IgG3 anti-MSP3 15 5049(±4) 11(±14) 3 10  23(±17) 9(±6) Control IgG3 15 0 11(±4)  9(±12) IFAT= endpoint titre of indirect immunofluorescence with P. falciparum clone3D7 mature schizonts infected RBCs as described; SGI % were the mean andSD in parenthesis of 3 different ADCI assays utilizing P. falciparumclone 3D7; DI % = percentage of direct inhibitory effect of antibodieson P. falciparum culture.

The 3 independent assays yielded the same results both upon the PaloAlto strain and the 3D7 clone with a stronger ADCI effect obtained withRAM1 IgG1 than RAM1 IgG3 (see Table II.). This set of in vitro studiesclearly establishes that RAM1 which binds to the MSP-3. b epitope canexert a parasite killing effect upon the main malaria speciesresponsible for the majority of deaths attributable to malaria, P.falciparum, in a strong and efficient manner, i.e. at least as strong asantibodies from Africans who have reached a state of protection whichcan be demonstrated by passive transfer of their immunoglobulins intonaive infected recipients. This biological effect was not only strong,but also obtained at very low concentrations of the recombinantantibody, i.e. at concentrations which correspond to only 5% of theconcentration of the polyclonal anti-MSP-3 antibodies in the African IgGprotective pool. As was the case with positive controls mentioned above,no direct effect upon merozoite invasion into red blood cells wasrecorded with RAM1 antibodies. Hence, the RAM1 recombinant antibodiesreproduced all the observations previously made with natural,polyclonal, protective antibodies.

In Vivo Studies in Immunocompromised Mice

As described above, in vivo studies may be performed in the mouse modeldescribed by Badell et al. (1995, 2000). The in vivo studies describedhere were performed using the newly developed immunocompromised mousemodel, in which human red blood cells can be grafted and a P. falciparumparasitemia obtained for durations of up to 4 and a half months. Thismodel is essentially an improvement over the initial descriptions madeby Badell et al. (1995) (see below) and correspond essentially to theprotocol described in Badell et al. (1995) (see below) and also Morenoet al. (2000) (see below). One of the major advantages of this P.falciparum animal model over primate models is that parasitemia remainspersistent at chronic level, i.e. at densities which are similar tothose obtained in human subjects. This contrasts with primate modelswhere parasitemia is fast rising and where the animals need to betreated within a few days, otherwise they will die of infection.Therefore, in the new mouse model, serial new mouse model, serialexperiments can be performed over several days or weeks and the effectsof various components investigated as of control antibodies can besequentially injected in the same animal which therefore constitute itsown control.

Mice: Male Beige/XID/Nude (BXN) mice of 6-8 weeks old were used. Theywere purchased from Charles River, USA, kept and manipulated inpathogens free conditions (sterile isolators and laminar flow hoods).

Immunomodulation of innate immunity in BXN mice: The number ofmacrophages and polymorphonuclears neutrophils (PMN) were respectivelyreduced by intraperitoneal injection (i.p.) of 0,2 ml liposomescontaining Cl₂MDP every 4-5 days (24) and of 300 μg of NIMP-R14monoclonal anti-PMN antibodies. The hybridoma producing NIMP-R14 MAb wasa gift from Dr. Malcolm Strath.

Parasites and culture: The P. falciparum African strains 3D7 and PaloAlto were used. Parasite blood stages were cultivated in RPMI 1640(Gibco BRL, Grand Island, N.Y.) supplemented with hypoxanthine 30 mM(RPMI-Hypox) and 0.5% Albumax (Gibco BRL, Grand Island, N.Y.) andsynchronized by repeated sorbitol treatment and by flotation onplasmagel (Plasmagel, BELLON, Neuilly-Sur-Seine, France).

Graft of human red blood cells (HuRBC) into the mice: Human blood fromAB+ blood donors with no history of malaria was collected byvenipuncture in either, CPD anticoagulant (MacoPharma, Tourcoing-France)or sodium heparin (Sanofi Winthrop, Gentilly-France) and spun at 600 g,10 min. at 20° C. After elimination of the white cells (buffy coat)packed red blood cells were suspended in Saline-Adenine-Glucose SAGM(MacoPharma, Tourcoing-France), and kept at 4° C. for a maximum of 30days. Before use, HuRBC were washed 3 times at 600 g with RPMI-1640(Gibco BRL, Grand Island, N.Y.) supplemented with hypoxanthine(RPMI-Hypox). 2 ml of washed HuRBC at 50% hematocrit were injected i.pevery 3-4 days to each mouse

Graft of human parasitised red blood cells (P.f.-HuRBC) into the mice:Highly synchronized ring forms (parasitaemia between 1-3%) were washed 5min. at 400 g in RPMI-Hypoxanthine. 2 ml of this suspension at 50%hematocrit in RPMI-Hypoxanthine were injected i.p into each mice,followed by repeated injection of non-infected hu-RBC every 3-4 days asmentioned above. Daily thin blood films drawn from the tail vein wereused to monitor the development of the parasitaemia.

Passive Transfer in P.f.-HuRBC-BXN Mouse Model

Human peripheral blood mononuclear cells (hu-PBMC): Similar criteria asmentioned above were used to collected blood. The total hu-PBMC cellswere isolated by ficoll-hypaque and washed twice with Hank's balancedsalt solution (HBSS) buffered with 35 mM of Hepes (Gibco, BRL). HuMNwere enriched by adherence to plastics. Attached monocytes were removedby cells scrapper and the number of monocytes determinate bynon-specific esterase (NSE) staining (mean 70%) and the viabilityestimated by trypan's blue (mean 85%) Either 3×10⁷ hu-PBMC or 3×10⁶purified HUMN were engrafted in each mouse.

Control IgG preparations: A pool of hyper-immune African IgG (PIAG) waspurified from 200 pooled sera from protected individuals living in theIvory Coast as described by Bouharoun-Tayoun et al. These subjects arereferred to as protected since they have reached a state of clinicalimmunity to malaria. A pool of control IgG from normal French blooddonors with no history of malaria was prepared following the sameprocedure. For passive transfer experiments, total HI-IgG were injectedat a dose of 200 mg/kg (6 mg/mouse).

Sampling of mice at regular intervals after IgG transfer anddetermination of antibody concentrations by ELISA showed that catabolismled to a progressive disappearance of the transferred antibodies within7 days.

Preparation of Anti-MSP3/b and Anti-RESA antibodies: The IgG were loadedon a Sulfolink column (Pierce, Rockford, Ill.) that was coated withMSP3/b-Cys peptide according to manufacturer's instruction, and run in aclosed circuit for 24 hours at 1 ml/10 min rate. After washing with PBSfor 18 hours, the specific IgG were eluted from the column with glycine0.2M, pH2.5 (Sigma, St-Louis, Mo.). 1 ml fractions were collected and pHadjusted to 5.0 with 2M Tris in NaOH (Sigma, St-Louis, Mo.) . BSA at 2mg/ml was added to conserve the biological activity of the antibodies(Boehringer, Manheim) . The reactivity of each fraction against thepeptide was monitored by ELISA, as previously described, and thosecontaining specific anti-MSP3/b antibodies were pooled and concentratedto 500 μl with centricon30 (Millipore, Bedford, Mass.). To avoiddegradation of those antibodies, 1% of bovine albumin (Sigma, St Louis,USA) is added immediately after concentration. Then, the antibodies weredialyzed against PBS and RPMI 1640, filtered and stored at 4° C. untilused. Anti-RESA antibodies were obtained following the same experimentalprocedure as described above. The peptide sequence used was:(H-(EENVEHDA)₂-(EENV)₂-OH) (SEQ ID NO: 86)

For passive transfer experiments, the amount of immunopurified anti MSP3or RESA antibodies was adjusted by IFAT titters and 250 μl/mice of thesolution was added each time.

Anti-RAM1 IgG1and IgG3 antibodies and control anti-Rh D antibody, wereinjected at a rate of 1.2 mg per Kilogramme of bodyweight, via IP route

Immunoassays: Titration of purified antibodies on native protein: Thetiter of each concentrated antibody preparation was determined byincubating serial doubling dilutions on air-dried thin blood smears ofthe NF54 strain. After it, an anti-human FITC-labeled secondary antibody(Biosys, France) diluted 1/200 in PBS and Evans blue 1/5000 (Sigma,St-Louis, Mo.) was added. The titer is the highest dilution giving apositive response. MSP3 antibodies were titered on slides prepared withcultures containing a high percentage of very mature schizonts. For RESAantibodies, cultures with a high percentage of ring forms were used.

Western Blot: The specificity of the antibodies was also tested onwestern blot of parasite extract on SDS-page separation and transfer onnitro-cellulose paper of NF-54 strain after plasmagel flotation. Theantibodies were diluted 1/100 and revealed with a secondary anti-humanIgG PAL-labeled diluted 1/7500 (Promega, Madison, Wis., USA) using theNBT/BCIP system for the color reaction (Promega, Madison, Wiss., USA).

RESULTS

In P. falciparum BXN mice, the inoculation of normal blood monocytes didnot alter significantly the course of parasitemia (see FIGS. 15 and 17).Similarly, the injection of control anti-RESA antibodies had no effectwhatsoever, either alone or following inoculation of normal humanmonocytes (FIG. 15). Similarly, anti-MSP-1 antibodies were found to haveno significant effect upon the course of parasitemia. Conversely, theinoculation of affinity-purified human anti-MSP-3 antibodies, eitherobtained by affinity-purification on the MSP-3.b synthetic peptide orobtained by affinity-purification on the recombinant C-terminus MSP-3recombinant cleared the parasitemia in BXN mice as fast as drugtreatment by the fastest active antimalarial drugs, such as chloroquineor artemisin (FIG. 16). It is noteworthy that in those experiments,anti-MSP-3 antibodies affinity purified on either the synthetic peptideor the recombinant antigen had a faster and more profound effect thantotal protected African adults IgG in which anti-MSP-3 antibodies wereat the same concentration as the purified ones (FIG. 18). In animalsreceiving either recombinant RAM1 IgG1 or recombinant RAM1 IgG3antibodies, the inoculation of normal blood monocytes alone had nosignificant effect upon the course of parasitemia (a significant exampleis shown in FIG. 19). In the same animals, the subsequent inoculation ofthe negative control anti-body, a recombinant antibody directed to theRhesus D antigen, had no significant effect (there was a slight decreaseover the first day which was followed by a re-increase in parasitemia tothe initlal level (FIG. 19) In contrast, the final inoculation ofrecombinant antibody RAM1 IgG1resulted in the fast and total clearanceof the P. falciparum circulating parasitemia in those humanised mice(FIG. 19). Similarly, the inoculation of recombinant antibody RAM1 IgG3also induced a profound decrease in parasitemia which, however, was nottotal. Therefore, the same difference between recombinant RAM1 IgG1 andRAM1 IgG3 observed under in vitro conditions was also observed under invivo conditions in humanized immunocompromised mice. It is noteworthythat the recombinant RAM1 IgG1 induces a decrease in parasitemia of thesame intensity as polyclonal affinity-purified human antibodies toMSP-3.b and that the clearance of parasitemia was total, i.e. resultedin an absence of parasitemia over 96 hours in similar manner asaffinity-purified polyclonal antibody to MSP-3 and in contrast to totalprotective African IgG.

Therefore, both in vitro and in vivo results produced convergent data todemonstrate the strong biological effect of recombinant RAM1 IgG1antibody and its ability to mediate P. falciparum killing in cooperationwith normal blood monocytes in the ADCI effect which is at the origin ofthe discovery of MSP-3 target antigen.

DISCUSSION

The strategy followed for construction of the library was to attempt toclone as many antibody genes as possible from the 13 malaria immuneindividuals from Senegal, Africa. This presents some importantadvantages as opposed to the strategy of trying to cover the entirerepertoire from only a single or a few individuals. Immunity to malariais supposed to be one of the major selective pressures exerted tomankind in malaria endemic areas during evolution. It has beenpostulated (Antibody repertoires and pathogen recognition: the role ofgermline diversity and somatic hypermutation. PhD thesis by MihaelaOprea, 1999, Santa Fe Institute) that an important factor in successfuldevelopment of malaria immunity in an individual is the possession of alimited number of selected heavy chain genes, e.g. 10⁵, encoding broadlyreactive antibodies. The role of such antibodies would be to interactwith all important and harmful antigens in order to initiate an antibodyresponse progressively leading to a higher quality response in one andthe same individual. The higher quality is manifested in specificity,affinity and protective biological effects. By cloning 5×10⁷ differentheavy chain genes from 13 individuals the library on the averagecontains 4×10⁶ different genes from each individual thus enhancing thelikelihood of having cloned at least one copy of each of the 10⁵important genes. The other strategy, relying on cloning a similar sizelibrary from one individual would at the most result in the cloning ofone full repertoire of the 10⁵ basic repertoire of antibodies.

The antibodies provided here provide a novel strategy for prevention andtreatment of malaria. If ordinary ADCC and phagocytosis were the solemechanisms at play a high initial parasitemia would lead to a largeconsumption of immunoglobulin and consequently to a lower fractionalreduction in parasitemia. Therefore it is believed that theantibody-merozoite complex stimulates the monocyte in acontact-dependent manner to secrete the effector substance(s)responsible for a subsequent non-contact-dependent inhibition of thegrowth of the parasite. Candidate molecules for the inhibitorysubstances are nitrogen oxide and TNF-α. Thus the high initialconcentration of parasites is assumed to induce a high and efficientconcentration of anti-parasitic substances leading to a high reductionrate.

Exploitation of the naturally occurring anti-parasitic host/pathogeninteraction (premunition, ADCI) is a novel way of developing atherapeutical anti-parasitic activity. The immunological mechanism thatis exploited by these antibodies is the co-operation between the threecomponents, merozoite—antibody—monocyte leading to inhibition ofparasite growth (ADCI) through secretion of TNF-α and other substances,e.g. nitrogen oxide species and oxygen radicals. This mechanism ofprotection requires only a minority of the merozoites to interactphysically with antibody and monocyte. Indeed, the majority of theparasites are only exposed to the substances released as a consequenceof the bystander merozoite-antibody-monocyte interaction.

The use of recombinant antibodies ensures that the appropriate isotypecan be used. This would not be the case if antibodies purified frominfected individuals were used, e.g. for the ADCI mechanism, IgG1 andIgG3 isotypes are important.

The sub-populations of target antigen-escape mutants will not have adirect survival advantage compared to the wild-type and therefore do notinfluence the general level of anti-parasitic activity. Only in thesituation where the number of wild-type parasites had been reduceddramatically would the monocyte stimulus drop. A minority of parasitespossessing wild-type target-antigen would be sufficient for stimulatingthe monocyte to release substances with anti-parasitic activity.Therefore, resistance to this anti-parasitic mechanism is a minorconcern. Studies of the conservation of the relevant domains of thetarget molecule MSP-3, have indeed s confirmed that immunologicalselection pressure seemingly is absent as a consequence of theabove-mentioned immunological mechanism (Stricker et al 1999).

Insensitivity of the parasites to the action of the substances releasedby monocytes and other effector cells does not seem to be a problem.This is substantiated by the clinical observation of life-longpersistence of the so-called premunition once it has been obtained andis maintained by continuous exposure to the parasite.

Resistance to this mechanism will be a minor concern because it mimicsand exploits indirect control mechanisms established by host/pathogenco-existence through thousands and thousands of generations. Therefore,these antibodies provide an anti-parasitic effect without the risk ofinduction of parasite resistance.

Use of recombinant antibodies circumvents the uncertainty connected withthe parameters influencing each step in the classical vaccinedevelopment process: Antigen, vaccine formulation, immunogenicity,individual immune response genes, immune response and finally, theinteraction of host/parasite/antibody.

DEPOSITS UNDER THE BUDAPEST TREATY

Deposits of E. coli TOP10 bacteria containing plasmids encoding RAM Fabfragments have been deposited under the terms of the Budapest Treaty asfollows:

Clone:

TOP10/F′Tet(R)/pFAB73H+RAM1

Depository Institution:

DSMZ-Deutsche Sammiung von Mikroorganismen und Zellkulturen GmbH,Mascheroder Weg 1 b, D-38124 Braunschweig, Germany.

Date of Deposit:

14 Aug. 2002

Accession Number:

DSM 15134

Depositor:

Morten Hanefeld Dziegiel, HS Blodbank, Copenhagen University Hospital,Blegdamsvej 9, DK-2100 Copenhagen, Denmark.

Clone:

TOP10/F′Tet(R)/pFAB73H+RAM2

Depository Institution:

DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH,Mascheroder Weg 1 b, D-38124 Braunschweig, Germany.

Date of Deposit:

14 Aug. 2002

Accession Number:

DSM 15135

Depositor:

Morten Hanefeld Dziegiel, HS Blodbank, Copenhagen University Hospital,Blegdamsvej 9, DK-2100 Copenhagen, Denmark.

Clone:

TOP10/F′Tet(R)/pFAB73H+RAM3

Depository Institution:

DSMZ-Deutsche Samnlung von Mikroorganismen und Zellkulturen GmbH,Mascheroder Weg 1 b, D-38124 Braunschweig, Germany.

Date of Deposit:

14 Aug. 2002

Accession Number:

DSM 15136

Depositor:

Morten Hanefeld Dziegiel, HS Blodbank, Copenhagen University Hospital,Blegdamsvej 9, DK-2100 Copenhagen, Denmark.

The vectors in each of these clones carry the Vk, Ck and VH genesencoding the relevant anti-MSP3 Fab as described above.

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1. A purified human antibody, or antigen binding fragment thereof,specific for the Merozoite Surface Protein-3 (MSP-3) antigen ofPlasmodium falciparum, wherein the antibody comprises: a) a heavy chainwith Complementarity Determining Regions (CDRs) having the amino acidsequences: SYAMH, which corresponds to amino acid residues 31-35 of SEQID NO: 1; VISYDGSNKYYADSVKG, which corresponds to amino acid residues50-66 of SEQ ID NO: 1; and GASS which corresponds to amino acid residues99-102 of SEQ ID NO: 1; and b) a light chain with CDRs having amino theamino sequences: RASQSISSWLA, which corresponds to amino acid residues24-34 of SEQ ID NO: 2; KASSLES, which corresponds to amino acid residues50-56 of SEQ ID NO: 2; and QQYKSFPYT, which corresponds to amino acidresidues 89-97 of SEQ ID NO:
 2. 2. A purified human antibody, or antigenbinding fragment thereof, specific for the Merozoite Surface Protein-3(MSP-3) antigen of Plasmodium falciparum, wherein the antibodycomprises: a) a heavy chain with Complementarity Determining Regions(CDRs) having the amino acid sequences: SYGMS, which corresponds toamino acid residues 31-35 of SEQ ID NO: 3; TISSGGSYTYYPDSVKG, whichcorresponds to amino acid residues 50-66 of SEQ ID NO: 3; andLYYGYRYYFDY, which corresponds to amino acid residues 99-109 of SEQ IDNO: 3; and b) a light chain with CDRs having amino the amino sequences:QASQDITNSLN, which corresponds to amino acid residues 24-34 of SEQ IDNO: 4; DAFTLKT, which corresponds to amino acid residues 50-56 of SEQ IDNO: 4; and QQSHRVPFT, which corresponds to amino acid residues 89-97 ofSEQ ID NO:
 4. 3. A purified human antibody, or antigen binding fragmentthereof, specific for the Merozoite Surface Protein-3 (MSP-3) antigen ofPlasmodium falciparum, wherein the antibody comprises: a) a heavy chainwith Complementarity Determining Regions (CDRs) having the amino acidsequences: SYAMH, which corresponds to amino acid residues 31-35 of SEQID NO: 5; VISYDGSNKYYADSVKG, which corresponds to amino acid residues50-66 of SEQ ID NO: 5; and DSGGIAARLGGYFDL, which corresponds to aminoacid residues 99-113 of SEQ ID NO: 5; and b) a light chain with CDRshaving amino the amino sequences: RASQGISSYLA, which corresponds toamino acid residues 24-34 of SEQ ID NO: 6; AASTLQS, which corresponds toamino acid residues 50-56 of SEQ ID NO: 6; and QQGPT, which correspondsto amino acid residues 89-93 of SEQ ID NO:
 6. 4. The antibody, orantigen binding fragment thereof, according to any of claims 1, 2, and3, which is specific for the Merozoite Surface Protein 3 (MSP-3)₁₉₄₋₂₅₇antigen.
 5. The antibody, or antigen binding fragment thereof, accordingto claim 4, which is specific for an epitope having the amino acidsequence ILGWEFGGGVP (SEQ ID NO: 10), which corresponds to residues220-230 of the Merozoite Surface Protein 3 (MSP-3) antigen.
 6. Theantibody, or antigen binding fragment thereof, according to claim 1comprising the VH domain of Recombinant Anti-MSP-3 No. 1 (RAM1) havingthe amino acid sequence of SEQ ID NO:
 1. 7. The antibody, or antigenbinding fragment thereof, according to claim 2 comprising the VH domainof Recombinant Anti-MSP-3 No. 2 (RAM2) having the amino acid sequence ofSEQ ID NO:
 3. 8. The antibody, or antigen binding fragment thereof,according to claim 3 comprising the VH domain of Recombinant Anti-MSP-3No. 3 (RAM3) having the amino acid sequence of SEQ ID NO:
 5. 9. Theantibody, or antigen binding fragment thereof, according to claim 1comprising the VL domain of Recombinant Anti-MSP-3 No. 1 (RAM1) havingthe amino acid sequence of SEQ ID NO:
 2. 10. The antibody, or antigenbinding fragment thereof, according to claim 2 comprising the VL domainof Recombinant Anti-MSP-3 No. 2 (RAM2) having the amino acid sequence ofSEQ ID NO:
 4. 11. The antibody, or antigen binding fragment thereof,according to claim 3 comprising the VL domain of Recombinant Anti-MSP-3No. 3 (RAM3) having the amino acid sequence of SEQ ID NO:
 6. 12. Theantibody, or antigen binding fragment thereof, according to claim 6further comprising the VL domain of Recombinant Anti-MSP-3 No. 1 (RAM1)having the amino acid sequence of SEQ ID NO:
 2. 13. The antibody, orantigen binding fragment thereof, according to claim 7 furthercomprising the VL domain of Recombinant Anti-MSP-3 No. 2 (RAM2) havingthe amino acid sequence of SEQ ID NO:
 4. 14. The antibody, or antigenbinding fragment thereof, according to claim 8 further comprising the VLdomain of Recombinant Anti-MSP-3 No. 3 (RAM3) having the amino acidsequence of SEQ ID NO:
 6. 15. The antibody, or antigen binding fragmentthereof, according to any one of claims 1, 2, and 3, which comprises aconstant region selected from: gamma 1; gamma 2; gamma 3; gamma 4; mu;alpha 1; alpha 2; delta; or epsilon isotypes.
 16. The antibody, orantigen binding fragment thereof, according to claim 15, wherein theconstant region is selected from: the gamma 1; gamma 2; gamma 3; orgamma 4 isotypes, to form an IgG molecule.
 17. The antibody, or antigenbinding fragment thereof, according to claim 16, wherein the constantregion is selected from: the gamma 1 or the gamma 3 isotypes, to form anIgG1 or IgG3 isotype.
 18. The antibody, or antigen binding fragmentthereof, according to claim 17 having the allotype G1m(a,z), G1m(f),G3m(b), G3m(c3c5), G3m(c3), or G3m(s).
 19. The antibody, or antigenbinding fragment thereof, according to claim 18 having the allotypeG1m(a,z) or G3m(b).
 20. A composition comprising the antibody, orantigen binding fragment thereof, according to any of claims 1, 2, and3, wherein the antibody or antigen binding fragment thereof binds to theMerozoite Surface Protein-3 (MSP-3) antigen of Plasmodium falciparum.21. The antibody, or antigen binding fragment thereof, of any of claims1, 2, and 3, wherein the antibody, or antigen binding fragment thereof,is monoclonal.
 22. The antibody, or antigen binding fragment thereof, ofany of claims 1, 2, and 3, wherein the antibody, or antigen bindingfragment thereof, is recombinant.
 23. A pharmaceutical compositioncomprising the antibody, or antigen binding fragment thereof, accordingto any of claims 1, 2, and
 3. 24. The pharmaceutical composition ofclaim 23, wherein the dose of antibody, or antigen binding fragmentthereof, is chosen from 10 μg to 10 mg antibody, or antigen bindingfragment thereof, per kg body weight, 1.5 mg to 3.5 mg antibody, orantigen binding fragment thereof, per kg body weight, 2 mg antibody, orantigen binding fragment thereof, per kg body weight, 0.1 μg to 3 mgantibody, or antigen binding fragment thereof, per kg body weight, 0.1mg to 2 mg antibody, or antigen binding fragment thereof, per kg bodyweight, 0.5 mg to 1.5 mg antibody, or antigen binding fragment thereof,per kg body weight, 0.75 to 1 mg antibody, or antigen binding fragmentthereof, per kg body weight, and 1 mg antibody, or antigen bindingfragment thereof, per 1 kg body weight.
 25. A method of diagnosis ofmalaria comprising taking a sample of bodily fluid from an individual,contacting the sample with an antibody, or antigen binding fragment,according to any one of claims 1, 2, and 3 and determining the bindingof the antibody or antigen binding fragment to the sample, therebydetermining the presence or absence of malaria in the sample.
 26. Aprocess of treatment of a malaria infection comprising administering toan infected patient a purified antibody or antigen binding fragmentaccording to claim 1, 2 or
 3. 27. A process of treatment of a malariainfection comprising administering to an infected patient thecomposition of claim 23.